Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory digestive diseases

ABSTRACT

The present invention refers to the use of protein kinase inhibitors and more specifically to the use of inhibitors of the protein kinase c-Jun amino terminal kinase, JNK inhibitor sequences, chimeric peptides, or of nucleic acids encoding same as well as pharmaceutical compositions containing same, for the treatment of non-chronic or chronic inflammatory digestive diseases, such as colitis, including e.g. Ulcerative colitis, Crohn&#39;s disease, diversion colitis, ischemic colitis, infectious colitis, fulminant colitis, chemical colitis, microscopic colitis, lymphocytic colitis, and atypical colitis, etc.

RELATED APPLICATIONS

The present application is a U.S. National Phase Application ofInternational Application No. PCT/EP2009/003936 (filed 2 Jun. 2009)which claims priority to International Application No. PCT/EP2008/004340(filed 30 May 2008) both of which are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

A computer readable text file, entitled“067802-5028_SequenceListing.txt,” created on or about Nov. 29, 2010with a file size of about 70 kb contains the sequence listing for thisapplication and is hereby incorporated by reference in its entirety.

The present invention refers to the use of protein kinase inhibitors andmore specifically to the use of inhibitors of the protein kinase c-Junamino terminal kinase, JNK inhibitor sequences, chimeric peptides, or ofnucleic acids encoding same as well as pharmaceutical compositionscontaining same, for the treatment of non-chronic or chronicinflammatory digestive diseases, such as colitis, including e.g.ulcerative colitis, Crohn's colitis, diversion colitis, ischemiccolitis, infectious colitis, fulminant colitis, chemical colitis,microscopic colitis, lymphocytic colitis, and atypical colitis, etc.

The number of digestive diseases, particularly of non-chronic andchronic digestive diseases has significantly increased during the lastdecades in Western Civilizations and represents a considerable challengefor their public health care system. Digestive diseases are diseasesthat pertain to the gastrointestinal tract. This includes diseases ofthe esophagus, stomach, first, second, third and fourth part of theduodenum, jejunum, ileum, the ileo-cecal complex, large intestine,(ascending, transverse and descending colon) sigmoid colon and rectum.Chronic inflammatory digestive diseases occur frequently and arecharacterized by an inflammation of the colon, such as colitis,including e.g. Colitis ulcerosa (ulcerative colitis), Morbus Crohn(Crohn's disease), diversion colitis, ischemic colitis, infectiouscolitis, fulminant colitis, chemical colitis, microscopic colitis,lymphocytic colitis, collageneous colitis, indeterminate colitis andatypical colitis, etc., wherein Colitis ulcerosa (ulcerative colitis)and Morbus Crohn (Crohn's disease) represent the two major chronicinflammatory digestive diseases and two major types of inflammatorybowel disease. Both, Morbus Crohn and Colitis ulcerosa, are diseases theoccurrence of which has rapidly increased during last decades. E.g., inGermany it is estimated that about 0.01% to about 0.1% of thepopulation, i.e. about 10 to 100 people from about 100.000, suffer fromMorbus Crohn or Colitis ulcerosa. Furthermore, about 1 to about 8incidences of Morbus Crohn or Colitis ulcerosa occur each year withincreasing rate. Women are affected sligthly more often by Morbus Crohnand the male:female ratio of Colitis ulcerosa is about 1:1. The peak ageof onset of Colitis ulcerosa and Morbus Chrohn is between 15 and 30years. The second peak occurs between ages of 60 and 80 years.

Colitis ulcerosa is a form of inflammatory bowel disease (IBD) and, as asubtype of colitis, a disease of the intestine, specifically of thelarge intestine or colon. The symptoms of Colitis ulcerosa typicallyinclude characteristic ulcers or open sores in the colon. Colitisulcerosa ususally starts in the rectum and spreads continually into theproximal sections of the large intestine or colon, wherein the uppergastrointestinal tract is normally not affected. The main symptoms ofactive disease is usually diarrhea mixed with blood, of gradual onset,wherein patients typically suffer from cramplike abdominal pain. Colitisulcerosa is associated with a variety of extraintestinal manifestations(dermatologic rheumatology, ocular and others). It is an intermittentdisease, with periods of exacerbated symptoms, and periods that arerelatively symptom-free. Although Colitis ulcerosa has no known cause,there is a presumed genetic component to susceptibility. Furthermore, itis assumed, that the disease may be triggered in a susceptible person byenvironmental factors. However, the symptoms of Colitis ulcerosa rarelydiminish on their own but rather require treatment to go into remission,particularly, when the disease switches into a chronic disease state.

Therapy of Colitis ulcerosa typically depends of the degree anddistribution of the disease and usually involves treatment withanti-inflammatory drugs, immunosupression, and biological therapytargeting specific components of the immune response. In disease stateswith minor or medium inflammatory activity (mild and moderate distalcolitis), typically if the disease is restricted to the rectum, a commontherapy usually includes administration of 5-aminosalicylates, such asPentasa® or Salofalk®. Alternatively, local medication usingsuppositories or enemas may be applied. In addition, administration ofsteroid containing medications including e.g. hydrocortison, Budenosid,Beclomethason (Betnesol®) or Prednison (Rectodelt®) may be used,especially in acute therapy. In disease states with high inflammatoryactivity, typically, if Colitis ulcerosa spreads into the proximalsections of the large intestine or colon, administration ofglucocorticoides, such as Beclomethason (Betnesol®) or Prednison(Rectodelt®) or derivatives thereof, using intraveneous injection oradministration in rectal or oral forms, or administration ofimmunosuppressiva, such as e.g. azathioprin, methothrexat (MTX) orcyclosporin A are typically used. In few cases, antibody therapy usingantibodies, e.g. against TNF-alpha (INFLIXIMAB) or anti-CD4, may beapplied. In cases of severe chronic disease states, which do not respondto medication therapy, a colorectomy, i.e. a partial or total removal ofthe large bowel through surgery, is occasionally necessary and isconsidered to be a cure for the disease in cases. These therapiestypically decrease the symptoms of an acute attack due to Colitisulcerosa. However, none of these therapies appears to allow effectiveand enduring cure of this disease.

Morbus Crohn, or Crohn's disease (also known as regional enteritis) is afurther important chronic inflammatory digestive disease. It is asubtype of a chronic, episodic, inflammatory bowel disease (IBD) thataffects the entire wall of the bowel and intestines. In contrast tocolitis ulcerosa Morbus Crohn can affect any part of the completegastrointestinal tract, and as a result, the symptoms of Morbus Crohnvary among afflicted individuals. The disease is characterized by areasof inflammation with areas of normal lining between in a symptom knownas skip lesions. The main gastrointestinal symptoms are abdominal pain,diarrhea, constipation, vomiting and weight loss or gain. Morbus Crohncan also cause complications outside of the gastrointestinal tract suchas skin rashes, arthritis, and inflammation of the eye. Morbus Crohnaffects between 400,000 and 600,000 people in North America (Loftus, E.V.; P. Schoenfeld, W. J. Sandborn (January 2002). “The epidemiology andnatural history of Crohn's disease in population-based patient cohortsfrom North America: a systematic review”. Alimentary Pharmacology &Therapeutics 16 (1): 51-60.). Prevalence estimates for Northern Europehave ranged from 27-48 per 100,000 (Bernstein, Charles N. (July 2006).“The Epidemiology of Inflammatory Bowel Disease in Canada: APopulation-Based Study”. The American Journal of Gastroenterology 101(7): 1559-1568). Furthermore, Morbus Crohn tends to present initially inthe teens and twenties, with another peak incidence in the fifties toseventies, although the disease can occur at any age (Hanauer, StephenB. (March 1996). “Inflammatory bowel disease”. New England Journal ofMedicine 334 (13): 841-848; Gopal, Latha; Senthil Nachimuthu (2006 May23). Chrohns Disease, eMedicine). The cause of Morbus Crohn is notknown, however, it is believed to be an autoimmune disease that isgenetically linked. The highest relative risk occurs in siblings,affecting females slightly more frequently, wherein smokers are threetimes more likely to get Morbus Crohn. A number of medical treatmentsare utilized with the goal of putting and keeping the disease inremission. Such medical treatments include, inter alia, 5-aminosalicylicacid (5-ASA) formulations (Pentasa® capsules, Asacol tablets, Lialdatablets, Rowasa retention enemas), steroid medications, theadministration of immunomodulators (such as e.g. azathioprine,mercaptopurine (6-MP), and methotrexate), and newer biologicalmedications, such as anti-TNAalpha antibodies (e.g. INFLIXIMAB® andADALIMUMAB®). Similarly as discussed above for Colitis ulcerosa thesetherapies typically decrease the symptoms of an acute attack due toMorbus Crohn. However, none of these therapies appears to alloweffective and long lasting cure of Morbus Crohn's disease (with theexception of anti-TNF-alpha, which, however, exhibits an increased riskof side effects).

Other forms of colitis include e.g. diversion colitis, ischemic colitis,infectious colitis, fulminant colitis, chemical colitis, microscopiccolitis, lymphocytic colitis, collageneous colitis, indeterminatecolitis and atypical colitis, etc. However, there is no effective andlong lasting cure without the risk of side effects for any of thesediseases. Accordingly there exists an ongoing urgent need in the art toprovide alternative or improved medicaments, which allow new andpreferably improved therapies of the above diseases.

The object of the present invention is thus to provide alternative orimproved therapies, which allow new and preferably improved cure ofnon-chronic or chronic (inflammatory) digestive diseases, such ascolitis, including e.g. ulcerative colitis, Crohn's colitis, diversioncolitis, ischemic colitis, infectious colitis, fulminant colitis,chemical disease, microscopic colitis, lymphocytic colitis, and atypicalcolitis, etc.

This object is solved by the use of a JNK inhibitor sequence comprisingless than 150 amino acids in length for the preparation of apharmaceutical composition for treating non-chronic or chronicinflammatory digestive diseases in a subject.

The term “non-chronic or chronic inflammatory digestive disease” as usedherein typically denotes non-chronic or chronic inflammatory diseasesthat pertain to the gastrointestinal tract. This includes diseases ofthe esophagus, stomach, first, second, third and fourth part of theduodenum, jejunum, ileum, the ileo-cecal complex, large intestine,(ascending, transverse and descending colon) sigmoid colon and rectum.Preferably included in this respect are chronic inflammatory digestivediseases, which are characterized by an inflammation of the colon, suchas colitis, including e.g. Colitis ulcerosa (ulcerative colitis), MorbusCrohn (Crohn's disease), diversion colitis, ischemic colitis, infectiouscolitis, fulminant colitis, chemical colitis, microscopic colitis,lymphocytic colitis, collageneous colitis, indeterminate colitis andatypical colitis, etc.

The present inventors surprisingly found, that such JNK inhibitorsequences are suitable for treating such chronic or non-chronicinflammatory digestive diseases in a subject. This was neither obviousnor suggested by the prior art, even though JNK inhibitor sequences ingeneral have been known from the art.

JNK is the abbreviation for “c-Jun amino terminal kinase”, which is amember of the stress-activated group of mitogen-activated protein (MAP)kinases. These kinases have been implicated in the control of cellgrowth and differentiation, and, more generally, in the response ofcells to environmental stimuli. The JNK signal transduction pathway isactivated in response to environmental stress and by the engagement ofseveral classes of cell surface receptors. These cell surface receptorscan include cytokine receptors, serpentine receptors and receptortyrosine kinases. In mammalian cells, JNK has been implicated inbiological processes such as oncogenic transformation and mediatingadaptive responses to environmental stress. JNK has also been associatedwith modulating immune responses, including maturation anddifferentiation of immune cells, as well effecting programmed cell deathin cells identified for destruction by the immune system. This uniqueproperty made JNK signaling a promising target for developingpharmacological intervention. However, up to now a pharmacologicaleffect of JNK inhibitor sequences has been shown only for a limitednumber of diseases, including several neurological disorders such asischemic stroke and Parkinson's disease, wherein such JNK inhibitorsequences may include upstream kinase inhibitors (for example,CEP-1347), small chemical inhibitors of JNK (SP600125 and AS601245),which directly affect kinase activity e.g. by competing with theATP-binding site of the protein kinase, and peptide inhibitors of theinteraction between JNK and its substrates (D-JNKI and I-JIP) (see e.g.Kuan et al., Current Drug Targets—CNS & Neurological Disorders, February2005, vol. 4, no. 1, pp. 63-67(5)). In this context, the upstream kinaseinhibitor CEP-1347 (KT7515) is a semisynthetic inhibitor of the mixedlineage kinase family. CEP-1347 (KT7515) promotes neuronal survival atdosages that inhibit activation of the c-Jun amino-terminal kinases(JNKs) in primary embryonic cultures and differentiated PC12 cells aftertrophic withdrawal and in mice treated with 1-methyl-4-phenyltetrahydropyridine. Further, CEP-1347 (KT7515) was observed to promotelong term-survival of cultured chick embryonic dorsal root ganglion,sympathetic, ciliary and motor neurons (see e.g. Borasio et al,Neuroreport. 9(7): 1435-1439, May 11 1998.). The small chemical JNKinhibitor SP600125 was found to reduce the levels of c-Junphosphorylation, to protect dopaminergic neurons from apoptosis, and topartly restore the level of dopamine in MPTP-induced PD in C57BU6N mice(Wang et al., Neurosci Res. 2004 February; 48(2); 195-202). Theseresults indicated that JNK pathway is the major mediator of theneurotoxic effects of MPTP in vivo and inhibiting JNK activity mayrepresent a new and effective strategy to treat PD. A further example ofsmall chemical inhibitors is the aforementioned JNK-Inhibitor AS601245.AS601245 inhibits the JNK signaling pathway and promotes cell survivalafter cerebral ischemia. In vivo, AS601245 provided significantprotection against the delayed loss of hippocampal CA1 neurons in agerbil model of transient global ischemia. This effect is mediated byJNK inhibition and therefore by c-Jun expression and phosphorylation(see e.g. Carboni et al., J Pharmacol Exp Ther. 2004 July; 310(1):25-32.Epub 2004 Feb. 26). However, summarizing the above, the shownpharmacological effects of those JNK inhibitor sequences only provedusability for a limited number of diseases, particularly severalneurological disorders such as ischemic stroke and Parkinson's disease.Thus, it was a surprising result, that JNK inhibitor sequences may beused for the treatment of non-chronic or chronic inflammatory digestivediseases.

In the context of the present invention, a INK inhibitor sequence asdefined above may be typically derived from a human or rat IB1 sequence,preferably from an amino acid sequence as defined or encoded by any ofsequences according to SEQ ID NO: 102 (depicts the IB1 cDNA sequencefrom rat and its predicted amino acid sequence), SEQ ID NO: 103 (depictsthe IB1 protein sequence from rat encoded by the exon-intron boundary ofthe rIB1 gene-splice donor), SEQ ID NO: 104 (depicts the IB1 proteinsequence from Homo sapiens), or SEQ ID NO: 105 (depicts the IB1 cDNAsequence from Homo sapiens), more preferably from an amino acid sequenceas defined or encoded by any of sequences according to SEQ ID NO: 104(depicts the IB1 protein sequence from Homo sapiens), or SEQ ID NO: 105(depicts the IB1 cDNA sequence from Homo sapiens), or from any fragmentsor variants thereof. In other words, the JNK inhibitor sequencecomprises a fragment, variant, or variant of such fragment of a human orrat IB1 sequence. Human or rat IB sequences are defined or encoded,respectively, by the sequences according to SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104 or SEQ ID NO: 105.

Preferably, such a JNK inhibitor sequence as used herein comprises atotal length of less than 150 amino acid residues, preferably a range of5 to 150 amino acid residues, more preferably 10 to 100 amino acidresidues, even more preferably 10 to 75 amino acid residues and mostpreferably a range of 10 to 50 amino acid residues, e.g. 10 to 30, 10 to20, or 10 to 15 amino acid residues.

More preferably, such a JNK inhibitor sequence and the above ranges maybe selected from any of the above mentioned sequences, even morepreferably from an amino acid sequence as defined according to SEQ IDNO: 104 or as encoded by SEQ ID NO: 105, even more preferably in theregion between nucleotides 420 and 980 of SEQ ID NO: 105 or amino acids105 and 291 of SEQ ID NO: 104, and most preferably in the region betweennucleotides 561 and 647 of SEQ ID NO: 105 or amino acids 152 and 180 ofSEQ ID NO: 104.

According to a particular embodiment, a JNK inhibitor sequence as usedherein typically binds JNK and/or inhibits the activation of at leastone JNK activated transcription factor, e.g. c-Jun or ATF2 (see e.g. SEQID NOs: 15 and 16, respectively) or Elk1.

Likewise, the JNK inhibitor sequence as used herein preferably comprisesor consists of at least one amino acid sequence according to any one ofSEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or a fragment, derivative orvariant thereof. More preferably, the JNK inhibitor sequence as usedherein may contain 1, 2, 3, 4 or even more copies of an amino acidsequence according to SEQ ID NOs: 1 to 4, 13 to 20 and 33 to 100, or avariant, fragment or derivative thereof. If present in more than onecopy, these amino acid sequences according to SEQ ID NOs: 1 to 4, 13 to20 and 33 to 100, or variants, fragments, or derivatives thereof as usedherein may be directly linked with each other without any linkersequence or via a linker sequence comprising 1 to 10, preferably 1 to 5amino acids. Amino acids forming the linker sequence are preferablyselected from glycine or proline as amino acid residues. Morepreferably, these amino acid sequences according to SEQ ID NOs: 1 to 4,13 to 20 and 33 to 100, or fragments, variants or derivatives thereof,as used herein, may be separated by each other by a hinge of two, threeor more proline residues.

The JNK inhibitor sequences as used herein may be composed of L-aminoacids, D-amino acids, or a combination of both. Preferably, the JNKinhibitor sequences as used herein comprise at least 1 or even 2,preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or 9 andeven more preferably at least 10 or more D- and/or L-amino acids,wherein the D- and/or L-amino acids may be arranged in the JNK inhibitorsequences as used herein in a blockwise, a non-blockwise or in analternate manner.

According to one preferred embodiment the JNK inhibitor sequences asused herein may be exclusively composed of L-amino acids. The JNKinhibitor sequences as used herein may then comprise or consist of atleast one “native JNK inhibitor sequence” according to SEQ ID NO: 1 or3. In this context, the term “native” or “native JNK inhibitorsequence(s)” is referred to non-altered JNK inhibitor sequencesaccording to any of SEQ ID NOs: 1 or 3, as used herein, entirelycomposed of L-amino acids.

Accordingly, the JNK inhibitor sequence as used herein may comprise orconsist of at least one (native) amino acid sequence NH₂—X_(n)^(b)—X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)—COOH (L-IB generic(s)) [SEQID NO: 3] and/or the JNK binding domain (JBDs) of IB1XRPTTLXLXXXXXXXQDS/TX (L-IB (generic)) [SEQ ID NO: 19]. In this context,each X typically represents an amino acid residue, preferably selectedfrom any (native) amino acid residue. X_(n) ^(a) typically representsone amino acid residue, preferably selected from any amino acid residueexcept serine or threonine, wherein n (the number of repetitions of X)is 0 or 1. Furthermore, each X_(n) ^(b) may be selected from any aminoacid residue, wherein n (the number of repetitions of X) is 0-5, 5-10,10-15, 15-20, 20-30 or more, provided that if n (the number ofrepetitions of X) is 0 for X_(n) ^(a), X_(n) ^(b) does preferably notcomprise a serine or threonine at its C-terminus, in order to avoid aserine or threonine at this position. Preferably, X_(n) ^(b) representsa contiguous stretch of peptide residues derived from SEQ ID NO: 1 or 3.X_(n) ^(a) and X_(n) ^(b) may represent either D or L amino acids.Additionally, the JNK inhibitor sequence as used herein may comprise orconsist of at least one (native) amino acid sequence selected from thegroup comprising the JNK binding domain of IB1 DTYRPKRPTTLNLFPQVPRSQDT(L-IB1) [SEQ ID NO: 17]. More preferably, the JNK inhibitor sequence asused herein further may comprise or consist of at least one (native)amino acid sequence NH₂-RPKRPTTLNLFPQVPRSQD-COOH (L-IB1(s)) [SEQ ID NO:1]. Furthermore, the JNK inhibitor sequence as used herein may compriseor consist of at least one (native) amino acid sequence selected fromthe group comprising the JNK binding domain of IB1 L-IB1 (s1)(NH₂-TLNLFPQVPRSQD-COOH, SEQ ID NO: 33); L-IB1(s2)(NH₂-TTLNLFPQVPRSQ-COOH, SEQ ID NO: 34); L-IB1(s3)(NH₂-PTTLNLFPQVPRS-COOH, SEQ ID NO: 35); L-IB1(s4)(NH₂-RPTTLNLFPQVPR-COOH, SEQ ID NO: 36); L-IB1(s5)(NH₂-KRPTTLNLFPQVP-COOH, SEQ ID NO: 37); L-IB1(s6)(NH₂-PKRPTTLNLFPQV-COOH, SEQ ID NO: 38); L-IB1(s7)(NH₂-RPKRPTTLNLFPQ-COOH, SEQ ID NO: 39); L-IB1(s8)(NH₂-LNLFPQVPRSQD-COOH, SEQ ID NO: 40); L-IB1(s9)(NH₂-TLNLFPQVPRSQ-COOH, SEQ ID NO: 41); L-IB1(s10)(NH₂-TTLNLFPQVPRS-COOH, SEQ ID NO: 42); L-IB1(s11)(NH₂-PTTLNLFPQVPR-COOH, SEQ ID NO: 43); L-IB1(s12)(NH₂-RPTTLNLFPQVP-COOH, SEQ ID NO: 44); L-IB1(s13)(NH₂-KRPTTLNLFPQV-COOH, SEQ ID NO: 45); L-IB1(s14)(NH₂-PKRPTTLNLFPQ-COOH, SEQ ID NO: 46); L-IB1(s15)(NH₂-RPKRPTTLNLFP-COOH, SEQ ID NO: 47); L-IB1(s16)(NH₂-NLFPQVPRSQD-COOH, SEQ ID NO: 48); L-IB1(s17) (NH₂-LNLFPQVPRSQ-COOH,SEQ ID NO: 49); L-IB1(s18) (NH₂-TLNLFPQVPRS-COOH, SEQ ID NO: 50);L-IB1(s19) (NH₂-TTLNLFPQVPR-COOH, SEQ ID NO: 51); L-IB1(s20)(NH₂-PTTLNLFPQVP-COOH, SEQ ID NO: 52); L-IB1(s21) (NH₂-RPTTLNLFPQV-COON,SEQ ID NO: 53); L-IB1(s22) (NH₂-KRPTTLNLFPQ-COOH, SEQ ID NO: 54);L-IB1(s23) (NH₂-PKRPTTLNLFP-COOH, SEQ ID NO: 55); L-IB1(s24)(NH₂-RPKRPTTLNLF-COOH, SEQ ID NO: 56); L-IB1(s25) (NH₂-LFPQVPRSQD-COOH,SEQ ID NO: 57); L-IB1(s26) (NH₂-NLFPQVPRSQ-COOH, SEQ ID NO: 58);L-IB1(s27) (NH₂-LNLFPQVPRS-COOH, SEQ ID NO: 59); L-IB1(s28)(NH₂-TLNLFPQVPR-COOH, SEQ ID NO: 60); L-IB1(s29) (NH₂-TTLNLFPQVP-COOH,SEQ ID NO: 61); L-IB1(s30) (NH₂-PTTLNLFPQV-COOH, SEQ ID NO: 62);L-IB1(s31) (NH₂—RPTTLNLFPQ-COOH, SEQ ID NO: 63); L-IB1(s32)(NH₂-KRPTTLNLFP-COOH, SEQ ID NO: 64); L-IB1(s33) (NH₂-PKRPTTLNLF-COOH,SEQ ID NO: 65); and L-IB1(s34) (NH₂-RPKRPTTLNL-COOH, SEQ ID NO: 66).

Additionally, the JNK inhibitor sequence as used herein may comprise orconsist of at least one (native) amino acid sequence selected from thegroup comprising the (long) JNK binding domain (JBDs) of IB1PGTGCGDTYRPKRPTTLNLFPQVPRSQDT (IB1-long) [SEQ ID NO: 13], the (long) INKbinding domain of IB2 IPSPSVEEPHKHRPTTLRLTTLGAQDS (IB2-long) [SEQ ID NO:14], the JNK binding domain of c-Jun GAYGYSNPKILKQSMTLNLADPVGNLKPH(c-Jun) [SEQ ID NO: 15], the JNK binding domain of ATF2TNEDHLAVHKHKHEMTLKFGPARNDSVIV (ATF2) [SEQ ID NO: 16] (see e.g. FIGS.1A-1C). In this context, an alignment revealed a partially conserved 8amino acid sequence (see e.g. FIG. 1A) and a further comparison of theJBDs of IB1 and IB2 revealed two blocks of seven and three amino acidsthat are highly conserved between the two sequences.

According to another preferred embodiment the JNK inhibitor sequences asused herein may be composed in part or exclusively of D-amino acids asdefined above. More preferably, these JNK inhibitor sequences composedof D-amino acids are non-native D retro-inverso sequences of the above(native) JNK inhibitor sequences. The term “retro-inverso sequences”refers to an isomer of a linear peptide sequence in which the directionof the sequence is reversed and the chirality of each amino acid residueis inverted (see e.g. Jameson et al., Nature, 368, 744-746 (1994); Bradyet al, Nature, 368, 692-693 (1994)). The advantage of combiningD-enantiomers and reverse synthesis is that the positions of carbonyland amino groups in each amide bond are exchanged, while the position ofthe side-chain groups at each alpha carbon is preserved. Unlessspecifically stated otherwise, it is presumed that any given L-aminoacid sequence or peptide as used according to the present invention maybe converted into an D retro-inverso sequence or peptide by synthesizinga reverse of the sequence or peptide for the corresponding nativeL-amino acid sequence or peptide.

The D retro-inverso sequences as used herein and as defined above have avariety of useful properties. For example, D retro-inverso sequences asused herein enter cells as efficiently as L-amino acid sequences as usedherein, whereas the D retro-inverso sequences as used herein are morestable than the corresponding L-amino acid sequences.

Accordingly, the JNK inhibitor sequences as used herein may comprise orconsist of at least one D retro-inverso sequence according to the aminoacid sequence NH₂—X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n) ^(b)—COOH(D-IB1 generic (s)) [SEQ ID NO: 4] and/or XS/TDQXXXXXXXLXLTTPRX (D-IB(generic)) [SEQ ID NO: 20]. As used in this context, X, X_(n) ^(a) andX_(n) ^(b) are as defined above (preferably, representing D aminoacids), wherein X_(n) ^(b) preferably represents a contiguous stretch ofresidues derived from SEQ ID NO: 2 or 4.

Additionally, the JNK inhibitor sequences as used herein may comprise orconsist of at least one D retro-inverso sequence according to the aminoacid sequence comprising the JNK binding domain (JBDs) of IB1TDQSRPVQPFLNLTTPRKPRYTD (D-IB1) [SEQ ID NO: 18]. More preferably, theJNK inhibitor sequences as used herein may comprise or consist of atleast one D retro-inverso sequence according to the amino acid sequenceNH₂-DQSRPVQPFLNLTTPRKPR-COOH (D-IB1(s)) [SEQ ID NO: 2]. Furthermore, theJNK inhibitor sequences as used herein may comprise or consist of atleast one D retro-inverso sequence according to the amino acid sequencecomprising the JNK binding domain (JBDs) of IB1 D-IB1(s1)(NH₂-QPFLNLTTPRKPR-COOH, SEQ ID NO: 67); D-IB1(s2)(NH₂-VQPFLNLTTPRKP-COOH, SEQ ID NO: 68); D-IB1(s3)(NH₂-PVQPFLNLTTPRK-COOH, SEQ ID NO: 69); D-IB1(s4)(NH₂-RPVQPFLNLTTPR-COOH, SEQ ID NO: 70); D-IB1(s5)(NH₂-SRPVQPFLNLTTP-COOH, SEQ ID NO: 71); D-IB1(s6)(NH₂-QSRPVQPFLNLTT-COOH, SEQ ID NO: 72); D-IB1(s7)(NH₂-DQSRPVQPFLNLT-COOH, SEQ ID NO: 73); D-IB1(s8)(NH₂—PFLNLTTPRKPR-COOH, SEQ ID NO: 74); D-IB1(s9)(NH₂-QPFLNLTTPRKP-COOH, SEQ ID NO: 75); D-IB1(s10)(NH₂-VQPFLNLTTPRK-COOH, SEQ ID NO: 76); D-IB1(s11)(NH₂-PVQPFLNLTTPR-COOH, SEQ ID NO: 77); D-IB1(s12)(NH₂-RPVQPFLNLTTP-COOH, SEQ ID NO: 78); D-IB1(s13)(NH₂-SRPVQPFLNLTT-COOH, SEQ ID NO: 79); D-IB1(s14)(NH₂-QSRPVQPFLNLT-COOH, SEQ ID NO: 80); D-IB1(s15)(NH₂-DQSRPVQPFLNL-COOH, SEQ ID NO: 81); D-IB1(s16)(NH₂-FLNLTTPRKPR-COOH, SEQ ID NO: 82); D-IB1(s17) (NH₂-PFLNLTTPRKP-COOH,SEQ ID NO: 83); D-IB1(s18) (NH₂-QPFLNLTTPRK-COOH, SEQ ID NO: 84);D-IB1(s19) (NH₂-VQPFLNLTTPR-COON, SEQ ID NO: 85); D-IB1(s20)(NH₂-PVQPFLNLTTP-COON, SEQ ID NO: 86); D-IB1(s21) (NH₂-RPVQPFLNLTT-COOH,SEQ ID NO: 87); D-IB1(s22) (NH₂-SRPVQPFLNLT-COOH, SEQ ID NO: 88); D-IB1(s23) (NH₂-QSRPVQPFLNL-COOH, SEQ ID NO: 89); D-IB1(s24)(NH₂-DQSRPVQPFLN-COOH, SEQ ID NO: 90); D-IB1(s25) (NH₂-DQSRPVQPFL-COOH,SEQ ID NO: 91); D-IB1(s26) (NH₂-QSRPVQPFLN-COOH, SEQ ID NO: 92);D-IB1(s27) (NH₂-SRPVQPFLNL-COOH, SEQ ID NO: 93); D-IB1(s28)(NH₂-RPVQPFLNLT-COOH, SEQ ID NO: 94); D-IB1(s29) (NH₂-PVQPFLNLTT-COOH,SEQ ID NO: 95); D-IB1(s30) (NH₂-VQPFLNLTTP-COOH, SEQ ID NO: 96);D-IB1(s31) (NH₂-QPFLNLTTPR-COOH, SEQ ID NO: 97); D-IB1(s32)(NH₂-PFLNLTTPRK-COOH, SEQ ID NO: 98); D-IB1(s33) (NH₂-FLNLTTPRKP-COOH,SEQ ID NO: 99); and D-IB1(s34) (NH₂-LNLTTPRKPR-COOH, SEQ ID NO: 100).

The JNK inhibitor sequences as used herein and as disclosed above arepresented in Table 1 (SEQ ID NO:s 1-4, 13-20 and 33-100). The tablepresents the name of the JNK inhibitor sequences as used herein, as wellas their sequence identifier number, their length, and amino acidsequence. Furthermore, Table 1 shows sequences as well as their genericformulas, e.g. for SEQ ID NO's: 1, 2, 5, 6, 9 and 11 and SEQ ID NO's: 3,4, 7, 8, 10 and 12, respectively. Table 1 furthermore discloses thechimeric sequences SEQ ID NOs: 9-12 and 23-32 (see below), L-IB1sequences SEQ ID NOs: 33 to 66 and D-IB1 sequences SEQ ID NOs: 67 to100.

TABLE 1 SEQUENCE/PEPTIDE SEQ NAME ID NO AA SEQUENCE L-IB1(s) 1 19RPKRPTTLNLFPQVPRSQD (NH₂-RPKRPTTLNLFPQVPRSQD-COOH) D-IB1 (s) 2 19DQSRPVQPFLNLTTPRKPR (NH₂-DQSRPVQPFLNLTTPRKPR-COOH) L-IB (generic) (s) 319 NH₂-X_(n) ^(b)-X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)-COOHD-IB (generic) (s) 4 19 NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n)^(b)-COOH L-TAT 5 10 GRKKRRQRRR (NH₂-GRKKRRQRRR-COOH) D-TAT 6 10RRRQRRKKRG (NH₂-RRRQRRKKRG-COOH) L-generic-TAT (s) 7 11 NH₂-X_(n)^(b)-RKKRRQRRR-X_(n) ^(b)-COOH D-generic-TAT (s) 8 11 NH₂-X_(n)^(b)-RRRQRRKKR-X_(n) ^(b)-COOH L-TAT-IB1(s) 9 31GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD(NH₂-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH) L-TAT-IB (generic) 10 29NH₂-X_(n) ^(b)-RKKRRQRRR-X_(n) ^(b)-X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n)^(b)-COOH (s) D-TAT-IB1(s) 11 31 DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG(NH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH) D-TAT-IB (generic) 12 29NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n) ^(b)-RRRQRRKKR-X_(n)^(b)-COOH (s) IB1-long 13 29 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT(NH₂-PGTGCGDTYRPKRPTTLNLFPQVPRSQDT-COOH) IB2-long 14 27IPSPSVEEPHKHRPTTLRLTTLGAQDS (NH₂-IPSPSVEEPHKHRPTTLRLTTLGAQDS-COOH) c-Jun15 29 GAYGYSNPKILKQSMTLNLADPVGNLKPH(NH₂-GAYGYSNPKILKQSMTLNLADPVGNLKPH-COOH) ATF2 16 29TNEDHLAVHKHKHEMTLKFGPARNDSVIV (NH₂-TNEDHLAVHKHKHEMTLKFGPARNDSVIV-COOH)L-IB1 17 23 DTYRPKRPTTLNLFPQVPRSQDT (NH₂-DTYRPKRPTTLNLFPQVPRSQDT-COOH)D-IB1 18 23 TDQSRPVQPFLNLTTPRKPRYTD (NH₂-TDQSRPVQPFLNLTTPRKPRYTD-COOH)L-IB (generic) 19 19 XRPTTLXLXXXXXXXQDS/TX(NH₂-XRPTTLXLXXXXXXXQDS/TX-COOH) D-IB (generic) 20 19XS/TDQXXXXXXXLXLTTPRX (NH₂-XS/TDQXXXXXXXLXLTTPRX-COOH) L-generic-TAT 2117 XXXXRKKRRQRRRXXXX (NH₂-XXXXRKKRRQRRRXXXX-COOH) D-generic-TAT 22 17XXXXRRRQRRKKRXXXX (NH₂-XXXXRRRQRRKKRXXXX-COOH) L-TAT-IB1 23 35GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT(NH₂-GRKKRRQRRRPPDTYRPKRPTTLNLFPQVPRSQDT-COOH) L-TAT-IB (generic) 24 42XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX (NH₂-XXXXXXXRKKRRQRRRXXXXXXXXRPTTLXLXXXXXXXQDS/TX- COOH) D-TAT-IB1 25 35TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG(NH₂-TDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG-COOH) D-TAT-IB (generic) 26 42XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX (NH₂-XT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX- COOH) L-TAT-IB1(s1) 27 30RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD (NH₂-RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH)L-TAT-IB1(s2) 28 30 GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD(NH₂-GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD-COOH) L-TAT-IB1(s3) 29 29RKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD (NH₂-RKKRRQRRRX_(n)^(c)RPKRPTTLNLFPQVPRSQD-COOH) D-TAT-IB1(s1) 30 30DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR (NH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR-COOH)D-TAT-IB1(s2) 31 30 DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG(NH₂-DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG-COOH) D-TAT-IB1(s3) 32 29DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKR (NH₂-DQSRPVQPFLNLTTPRKPRX_(n)^(c)RRRQRRKKR-COOH) L-IB1(s1) 33 13 TLNLFPQVPRSQD(NH₂-TLNLFPQVPRSQD-COOH) L-IB1(s2) 34 13 TTLNLFPQVPRSQ(NH₂-TTLNLFPQVPRSQ-COOH) L-IB1(s3) 35 13 PTTLNLFPQVPRS(NH₂-PTTLNLFPQVPRS-COOH) L-IB1(s4) 36 13 RPTTLNLFPQVPR(NH₂-RPTTLNLFPQVPR-COOH) L-IB1(s5) 37 13 KRPTTLNLFPQVP(NH₂-KRPTTLNLFPQVP-COOH) L-IB1(s6) 38 13 PKRPTTLNLFPQV(NH₂-PKRPTTLNLFPQV-COOH) L-IB1(s7) 39 13 RPKRPTTLNLFPQ(NH₂-RPKRPTTLNLFPQ-COOH) L-IB1(s8) 40 12 LNLFPQVPRSQD(NH₂-LNLFPQVPRSQD-COOH) L-IB1(s9) 41 12 TLNLFPQVPRSQ(NH₂-TLNLFPQVPRSQ-COOH) L-IB1(s10) 42 12 TTLNLFPQVPRS(NH₂-TTLNLFPQVPRS-COOH) L-IB1(s11) 43 12 PTTLNLFPQVPR(NH₂-PTTLNLFPQVPR-COOH) L-IB1(s12) 44 12 RPTTLNLFPQVP(NH₂-RPTTLNLFPQVP-COOH) L-IB1(s13) 45 12 KRPTTLNLFPQV(NH₂-KRPTTLNLFPQV-COOH) L-IB1(s14) 46 12 PKRPTTLNLFPQ(NH₂-PKRPTTLNLFPQ-COOH) L-IB1(s15) 47 12 RPKRPTTLNLFP(NH₂-RPKRPTTLNLFP-COOH) L-IB1(s16) 48 11 NLFPQVPRSQD(NH₂-NLFPQVPRSQD-COOH) L-IB1(s17) 49 11 LNLFPQVPRSQ(NH₂-LNLFPQVPRSQ-COOH) L-IB1(s18) 50 11 TLNLFPQVPRS(NH₂-TLNLFPQVPRS-COOH) L-IB1(s19) 51 11 TTLNLFPQVPR(NH₂-TTLNLFPQVPR-COOH) L-IB1(s20) 52 11 PTTLNLFPQVP(NH₂-PTTLNLFPQVP-COOH) L-IB1(s21) 53 11 RPTTLNLFPQV(NH₂-RPTTLNLFPQV-COOH) L-IB1(s22) 54 11 KRPTTLNLFPQ(NH₂-KRPTTLNLFPQ-COOH) L-IB1(s23) 55 11 PKRPTTLNLFP(NH₂-PKRPTTLNLFP-COOH) L-IB1(s24) 56 11 RPKRPTTLNLF(NH₂-RPKRPTTLNLF-COOH) L-IB1(s25) 57 10 LFPQVPRSQD (NH₂-LFPQVPRSQD-COOH)L-I81(s26) 58 10 NLFPQVPRSQ (NH₂-NLFPQVPRSQ-COOH) L-IB1(s27) 59 10LNLFPQVPRS (NH₂-LNLFPQVPRS-COOH) L-IB1(s28) 60 10 TLNLFPQVPR(NH₂-TLNLFPQVPR-COOH) L-IB1(s29) 61 10 TTLNLFPQVP (NH₂-TTLNLFPQVP-COOH)L-IB1(s30) 62 10 PTTLNLFPQV (NH₂-PTTLNLFPQV-COOH) L-IB1(s31) 63 10RPTTLNLFPQ (NH₂-RPTTLNLFPQ-COOH) L-IB1(s32) 64 10 KRPTTLNLFP(NH₂-KRPTTLNLFP-COOH) L-IB1(s33) 65 10 PKRPTTLNLF (NH₂-PKRPTTLNLF-COOH)L-IB1(s34) 66 10 RPKRPTTLNL (NH₂-RPKRPTTLNL-COOH) D-IB1(s1) 67 13QPFLNLTTPRKPR (NH₂-QPFLNLTTPRKPR-COOH) D-IB1(s2) 68 13 VQPFLNLTTPRKP(NH₂-VQPFLNLTTPRKP-COOH) D-IB1(s3) 69 13 PVQPFLNLTTPRK(NH₂-PVQPFLNLTTPRK-COOH) D-IB1(s4) 70 13 RPVQPFLNLTTPR(NH₂-RPVQPFLNLTTPR-COOH) D-IB1(s5) 71 13 SRPVQPFLNLTTP(NH₂-SRPVQPFLNLTTP-COOH) D-IB1(s6) 72 13 QSRPVQPFLNLTT(NH₂-QSRPVQPFLNLTT-COOH) D-IB1(s7) 73 13 DQSRPVQPFLNLT(NH₂-DQSRPVQPFLNLT-COOH) D-IB1(s8) 74 12 PFLNLTTPRKPR(NH₂-PFLNLTTPRKPR-COOH) D-IB1(s9) 75 12 QPFLNLTTPRKP(NH₂-QPFLNLTTPRKP-COOH) D-IB1(s10) 76 12 VQPFLNLTTPRK(NH₂-VQPFLNLTTPRK-COOH) D-IB1(s11) 77 12 PVQPFLNLTTPR(NH₂-PVQPFLNLTTPR-COOH) D-IB1(s12) 78 12 RPVQPFLNLTTP(NH₂-RPVQPFLNLTTP-COOH) D-IB1(s13) 79 12 SRPVQPFLNLTT(NH₂-SRPVQPFLNLTT-COOH) D-IB1(s14) 80 12 QSRPVQPFLNLT(NH₂-QSRPVQPFLNLT-COOH) D-IB1(s15) 81 12 DQSRPVQPFLNL(NH₂-DQSRPVQPFLNL-COOH) D-IB1(s16) 82 11 FLNLTTPRKPR(NH₂-FLNLTTPRKPR-COOH) D-IB1(s17) 83 11 PFLNLTTPRKP(NH₂-PFLNLTTPRKP-COOH) D-IB1(s18) 84 11 QPFLNLTTPRK(NH₂-QPFLNLTTPRK-COOH) D-IB1(s19) 85 11 VQPFLNLTTPR(NH₂-VQPFLNLTTPR-COOH) D-IB1(s20) 86 11 PVQPFLNLTTP(NH₂-PVQPFLNLTTP-COOH) D-IB1(s21) 87 11 RPVQPFLNLTT(NH₂-RPVQPFLNLTT-COOH) D-IB1(s22) 88 11 SRPVQPFLNLT(NH₂-SRPVQPFLNLT-COOH) D-IB1(s23) 89 11 QSRPVQPFLNL(NH₂-QSRPVQPFLNL-COOH) D-IB1(s24) 90 11 DQSRPVQPFLN(NH₂-DQSRPVQPFLN-COOH) D-IB1(s25) 91 10 DQSRPVQPFL (NH₂-DQSRPVQPFL-COOH)D-IB1(s26) 92 10 QSRPVQPFLN (NH₂-QSRPVQPFLN-COOH) D-IB1(s27) 93 10SRPVQPFLNL (NH₂-SRPVQPFLNL-COOH) D-IB1(s28) 94 10 RPVQPFLNLT(NH₂-RPVQPFLNLT-COOH) D-IB1(s29) 95 10 PVQPFLNLTT (NH₂-PVQPFLNLTT-COOH)D-IB1(s30) 96 10 VQPFLNLTTP (NH₂-VQPFLNLTTP-COOH) D-IB1(s31) 97 10QPFLNLTTPR (NH₂-QPFLNLTTPR-COOH) D-IB1(s32) 98 10 PFLNLTTPRK(NH₂-PFLNLTTPRK-COOH) D-IB1(s33) 99 10 FLNLTTPRKP (NH₂-FLNLTTPRKP-COOH)D-IB1(s34) 100 10 LNLTTPRKPR (NH₂-LNLTTPRKPR-COOH)

According to another preferred embodiment, the JNK inhibitor sequence asused herein comprises or consists of at least one variant, fragmentand/or derivative of the above defined native or non-native amino acidsequences according to SEQ ID NOs: 1-4, 13-20 and 33-100. Preferably,these variants, fragments and/or derivatives retain biological activityof the above disclosed native or non-native JNK inhibitor sequences asused herein, particularly of native or non-native amino acid sequencesaccording to SEQ ID NOs: 1-4, 13-20 and 33-100, i.e. binding JNK and/orinhibiting the activation of at least one JNK activated transcriptionfactor, e.g. c-Jun, ATF2 or Elk1. Functionality may be tested by varioustests, e.g. binding tests of the peptide to its target molecule or bybiophysical methods, e.g. spectroscopy, computer modeling, structuralanalysis, etc. Particularly, an JNK inhibitor sequence or variants,fragments and/or derivatives thereof as defined above may be analyzed byhydrophilicity analysis (see e.g. Hopp and Woods, 1981. Proc Natl AcadSci USA 78: 3824-3828) that can be utilized to identify the hydrophobicand hydrophilic regions of the peptides, thus aiding in the design ofsubstrates for experimental manipulation, such as in bindingexperiments, or for antibody synthesis. Secondary structural analysismay also be performed to identify regions of an JNK inhibitor sequenceor of variants, fragments and/or derivatives thereof as used herein thatassume specific structural motifs (see e.g. Chou and Fasman, 1974,Biochem 13: 222-223). Manipulation, translation, secondary structureprediction, hydrophilicity and hydrophobicity profiles, open readingframe prediction and plotting, and determination of sequence homologiescan be accomplished using computer software programs available in theart. Other methods of structural analysis include, e.g. X-raycrystallography (see e.g. Engstrom, 1974. Biochem Exp Biol 11: 7-13),mass spectroscopy and gas chromatography (see e.g. METHODS IN PROTEINSCIENCE, 1997, 1. Wiley and Sons, New York, N.Y.) and computer modeling(see e.g. Fletterick and Zoller, eds., 1986. Computer Graphics andMolecular Modeling, In: CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) may alsobe employed.

Accordingly, the INK inhibitor sequence as used herein may comprise orconsist of at least one variant of (native or non-native) amino acidsequences according to SEQ ID NOs: 1-4, 13-20 and 33-100. In the contextof the present invention, a “variant of a (native or non-native) aminoacid sequence according to SEQ ID NOs: 1-4, 13-20 and 33-100” ispreferably a sequence derived from any of the sequences according to SEQID NOs: 1-4, 13-20 and 33-100, wherein the variant comprises amino acidalterations of the amino acid sequences according to SEQ ID NOs: 1-4,13-20 and 33-100. Such alterations typically comprise 1 to 20,preferably 1 to 10 and more preferably 1 to 5 substitutions, additionsand/or deletions of amino acids according to SEQ ID NOs: 1-4, 13-20 and33-100, wherein the variant exhibits a sequence identity with any of thesequences according to SEQ ID NOs: 1-4, 13-20 and 33-100 of at leastabout 30%, 50%, 70%, 80%, 90%, 95%, 98% or even 99%.

If variants of (native or non-native) amino acid sequences according toSEQ ID NOs: 1-4, 13-20 and 33-100 as defined above and used herein areobtained by substitution of specific amino acids, such substitutionspreferably comprise conservative amino acid substitutions. Conservativeamino acid substitutions may include synonymous amino acid residueswithin a group which have sufficiently similar physicochemicalproperties, so that a substitution between members of the group willpreserve the biological activity of the molecule (see e.g. Grantham, R.(1974), Science 185, 862-864). It is evident to the skilled person thatamino acids may also be inserted and/or deleted in the above-definedsequences without altering their function, particularly if theinsertions and/or deletions only involve a few amino acids, e.g. lessthan twenty, and preferably less than ten, and do not remove or displaceamino acids which are critical to functional activity. Moreover,substitutions shall be avoided in variants as used herein, which lead toadditional threonines at amino acid positions which are accessible for aphosphorylase, preferably a kinase, in order to avoid inactivation ofthe JNK-inhibitor sequence as used herein or of the chimeric peptide asused herein in vivo or in vitro.

Preferably, synonymous amino acid residues, which are classified intothe same groups and are typically exchangeable by conservative aminoacid substitutions, are defined in Table 2.

TABLE 2 Preferred Groups of Synonymous Amino Acid Residues Amino AcidSynonymous Residue Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, HisLeu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, (Thr), Pro Thr Pro, Ser,Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile,Leu, Val Gly Ala, (Thr), Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, IlePhe Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu,Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys,Asn, His, (Thr), Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg,Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe,Ile, Val, Leu, Met Trp Trp

A specific form of a variant of SEQ ID NOs: 1-4, 13-20 and 33-100 asused herein is a fragment of the (native or non-native) amino acidsequences according to SEQ ID NOs: 1, 1-4, 13-20 and 33-100” as usedherein, which is typically altered by at least one deletion as comparedto SEQ ID NOs 1-4, 13-20 and 33-100. Preferably, a fragment comprises atleast 4 contiguous amino acids of any of SEQ ID NOs: 1-4, 13-20 and33-100, a length typically sufficient to allow for specific recognitionof an epitope from any of these sequences. Even more preferably, thefragment comprises 4 to 18, 4 to 15, or most preferably 4 to 10contiguous amino acids of any of SEQ ID NOs: 1-4, 13-20 and 33-100,wherein the lower limit of the range may be 4, or 5, 6, 7, 8, 9, or 10.Deleted amino acids may occur at any position of SEQ ID NOs: 1-4, 13-20and 33-100, preferably N- or C-terminally.

Furthermore, a fragment of the (native or non-native) amino acidsequences according to SEQ ID NOs: 1-4, 13-20 and 33-100, as describedabove, may be defined as a sequence sharing a sequence identity with anyof the sequences according to SEQ ID NOs: 1-4, 13-20 and 33-100 as usedherein of at least about 30%, 50%, 70%, 80%, 90%, 95%, 98%, or even 99%.

The JNK inhibitor sequences as used herein may further comprise orconsist of at least one derivative of (native or non-native) amino acidsequences according to SEQ ID NOs: 1-4, 13-20 and 33-100 as definedabove. In this context, a “derivative of an (native or non-native) aminoacid sequence according to SEQ ID NOs: 1-4, 13-20 and 33-100” ispreferably an amino acid sequence derived from any of the sequencesaccording to SEQ ID NOs: 1-4, 13-20 and 33-100, wherein the derivativecomprises at least one modified L- or D-amino acid (forming non-naturalamino acid(s)), preferably 1 to 20, more preferably 1 to 10, and evenmore preferably 1 to 5 modified L- or D-amino acids. Derivatives ofvariants or fragments also fall under the scope of the presentinvention.

“A modified amino acid” in this respect may be any amino acid which isaltered e.g. by different glycosylation in various organisms, byphosphorylation or by labeling specific amino acids. Such a label isthen typically selected from the group of labels comprising:

-   -   (i) radioactive labels, i.e. radioactive phosphorylation or a        radioactive label with sulphur, hydrogen, carbon, nitrogen,        etc.;    -   (ii) colored dyes (e.g. digoxygenin, etc.);    -   (iii) fluorescent groups (e.g. fluorescein, etc.);    -   (iv) chemoluminescent groups;    -   (v) groups for immobilization on a solid phase (e.g. His-tag,        biotin, strep-tag, flag-tag, antibodies, antigen, etc.); and    -   (vi) a combination of labels of two or more of the labels        mentioned under (i) to (v).

In the above context, an amino acid sequence having a sequence “sharinga sequence identity” of at least, for example, 95% to a query amino acidsequence of the present invention, is intended to mean that the sequenceof the subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted.

For sequences without exact correspondence, a “% identity” of a firstsequence may be determined with respect to a second sequence. Ingeneral, these two sequences to be compared are aligned to give amaximum correlation between the sequences. This may include inserting“gaps” in either one or both sequences, to enhance the degree ofalignment. A % identity may then be determined over the whole length ofeach of the sequences being compared (so-called global alignment), thatis particularly suitable for sequences of the same or similar length, orover shorter, defined lengths (so-called local alignment), that is moresuitable for sequences of unequal length.

Methods for comparing the identity and homology of two or moresequences, particularly as used herein, are well known in the art. Thusfor instance, programs available in the Wisconsin Sequence AnalysisPackage, version 9.1 (Devereux et at, 1984, Nucleic Acids Res. 12,387-395.), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identityand the % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of (Smith and Waterman (1981), J. Mol. Biol.147, 195-197.) and finds the best single region of similarity betweentwo sequences. Other programs for determining identity and/or similaritybetween sequences are also known in the art, for instance the BLASTfamily of programs (Altschul et at, 1990, J. Mol. Biol. 215, 403-410),accessible through the home page of the NCBI at world wide web sitencbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 183,63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U.S. A 85,2444-2448.).

JNK-inhibitor sequences as used according to the present invention andas defined above may be obtained or produced by methods well-known inthe art, e.g. by chemical synthesis or by genetic engineering methods asdiscussed below. For example, a peptide corresponding to a portion of anJNK inhibitor sequence as used herein including a desired region of saidJNK inhibitor sequence, or that mediates the desired activity in vitroor in vivo, may be synthesized by use of a peptide synthesizer.

JNK inhibitor sequence as used herein and as defined above, may befurthermore be modified by a trafficking sequence, allowing the JNKinhibitor sequence as used herein and as defined above to be transportedeffectively into the cells. Such modified JNK inhibitor sequence arepreferably provided and used as chimeric sequences.

According to a second aspect the present invention therefore providesthe use of a chimeric peptide including at least one first domain and atleast one second domain, for the preparation of a pharmaceuticalcomposition for treating non-chronic or chronic inflammatory digestivediseases in a subject, wherein the first domain of the chimeric peptidecomprises a trafficking sequence, while the second domain of thechimeric peptide comprises an JNK inhibitor sequence as defined above,preferably of any of sequences according to SEQ ID NO: 1-4, 13-20 and33-100 or a derivative or a fragment thereof.

Typically, chimeric peptides as used according to the present inventionhave a length of at least 25 amino acid residues, e.g. 25 to 250 aminoacid residues, more preferably 25 to 200 amino acid residues, even morepreferably 25 to 150 amino acid residues, 25 to 100 and most preferablyamino acid 25 to 50 amino acid residues.

As a first domain the chimeric peptide as used herein preferablycomprises a trafficking sequence, which is typically selected from anysequence of amino acids that directs a peptide (in which it is present)to a desired cellular destination. Thus, the trafficking sequence, asused herein, typically directs the peptide across the plasma membrane,e.g. from outside the cell, through the plasma membrane, and into thecytoplasm. Alternatively, or in addition, the trafficking sequence maydirect the peptide to a desired location within the cell, e.g. thenucleus, the ribosome, the endoplasmic reticulum (ER), a lysosome, orperoxisome, by e.g. combining two components (e.g. a component for cellpermeability and a component for nuclear location) or by one singlecomponent having e.g. properties of cell membrane transport and targetede.g. intranuclear transport. The trafficking sequence may additionallycomprise another component, which is capable of binding a cytoplasmiccomponent or any other component or compartment of the cell (e.g.endoplasmic reticulum, mitochondria, gloom apparatus, lysosomalvesicles). Accordingly, e.g. the trafficking sequence of the firstdomain and the JNK inhibitor sequence of the second domain may belocalized in the cytoplasm or any other compartment of the cell. Thisallows to determine localization of the chimeric peptide in the cellupon uptake.

Preferably, the trafficking sequence (being included in the first domainof the chimeric peptide as used herein) has a length of 5 to 150 aminoacid sequences, more preferably a length of 5 to 100 and most preferablya length of from 5 to 50, 5 to 30 or even 5 to 15 amino acids.

More preferably, the trafficking sequence (contained in the first domainof the chimeric peptide as used herein) may occur as a continuous aminoacid sequence stretch in the first domain. Alternatively, thetrafficking sequence in the first domain may be splitted into two ormore fragments, wherein all of these fragments resemble the entiretrafficking sequence and may be separated from each other by 1 to 10,preferably 1 to 5 amino acids, provided that the trafficking sequence assuch retains its carrier properties as disclosed above. These aminoacids separating the fragments of the trafficking sequence may e.g. beselected from amino acid sequences differing from the traffickingsequence. Alternatively, the first domain may contain a traffickingsequence composed of more than one component, each component with itsown function for the transport of the cargo JNK inhibitor sequence ofthe second domain to e.g. a specific cell compartment.

The trafficking sequence as defined above may be composed of L-aminoacids, D-amino acids, or a combination of both. Preferably, thetrafficking sequences (being included in the first domain of thechimeric peptide as used herein) may comprise at least 1 or even 2,preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or 9 andeven more preferably at least 10 or more D- and/or L-amino acids,wherein the D- and/or L-amino acids may be arranged in the JNKtrafficking sequences in a blockwise, a non-blockwise or in an alternatemanner.

According to one alternative embodiment, the trafficking sequence of thechimeric peptide as used herein may be exclusively composed of L-aminoacids. More preferably, the trafficking sequence of the chimeric peptideas used herein comprises or consists of at least one “native”trafficking sequence as defined above. In this context, the term“native” is referred to non-altered trafficking sequences, entirelycomposed of L-amino acids.

According to another alternative embodiment the trafficking sequence ofthe chimeric peptide as used herein may be exclusively composed ofD-amino acids. More preferably, the trafficking sequence of the chimericpeptide as used herein may comprise a D retro-inverso peptide of thesequences as presented above.

The trafficking sequence of the first domain of the chimeric peptide asused herein may be obtained from naturally occurring sources or can beproduced by using genetic engineering techniques or chemical synthesis(see e.g. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecularcloning: A laboratory manual. 2nd edition. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Sources for the trafficking sequence of the first domain may be employedincluding, e.g. native proteins such as e.g. the TAT protein (e.g. asdescribed in U.S. Pat. Nos. 5,804,604 and 5,674,980, each of thesereferences being incorporated herein by reference), VP22 (described ine.g. WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)),non-viral proteins (Jackson et al, Proc. Natl. Acad. Sci. USA 89:10691-10695 (1992)), trafficking sequences derived from Antennapedia(e.g. the antennapedia carrier sequence) or from basic peptides, e.g.peptides having a length of 5 to 15 amino acids, preferably 10 to 12amino acids and comprising at least 80%, more preferably 85% or even 90%basic amino acids, such as e.g. arginine, lysine and/or histidine.Furthermore, variants, fragments and derivatives of one of the nativeproteins used as trafficking sequences are disclosed herewith. Withregard to variants, fragments and derivatives it is referred to thedefinition given above for JNK inhibitor sequences as used herein.Variants, fragments as well as derivatives are correspondingly definedas set forth above for JNK inhibitor sequences as used herein.Particularly, in the context of the trafficking sequence, a variant orfragment or derivative may be defined as a sequence sharing a sequenceidentity with one of the native proteins used as trafficking sequencesas defined above of at least about 30%, 50%, 70%, 80%, 90%, 95%, 98%, oreven 99%.

In a preferred embodiment of the chimeric peptide as used herein, thetrafficking sequence of the first domain comprises or consists of asequence derived from the human immunodeficiency virus (HIV)1 TATprotein, particularly some or all of the 86 amino acids that make up theTAT protein.

For a trafficking sequence (being included in the first domain of thechimeric peptide as used herein), partial sequences of the full-lengthTAT protein may be used forming a functionally effective fragment of aTAT protein, i.e. a TAT peptide that includes the region that mediatesentry and uptake into cells. As to whether such a sequence is afunctionally effective fragment of the TAT protein can be determinedusing known techniques (see e.g. Franked et al, Proc. Natl. Acad. Sci,USA 86: 7397-7401 (1989)). Thus, the trafficking sequence in the firstdomain of the chimeric peptide as used herein may be derived from afunctionally effective fragment or portion of a TAT protein sequencethat comprises less than 86 amino acids, and which exhibits uptake intocells, and optionally the uptake into the cell nucleus. More preferably,partial sequences (fragments) of TAT to be used as carrier to mediatepermeation of the chimeric peptide across the cell membrane, areintended to comprise the basic region (amino acids 48 to 57 or 49 to 57)of full-length TAT.

According to a more preferred embodiment, the trafficking sequence(being included in the first domain of the chimeric peptide as usedherein) may comprise or consist of an amino acid sequence containing TATresidues 48-57 or 49 to 57, and most preferably a generic TAT sequenceNH₂—X_(n) ^(b)-RKKRRQRRR-X_(n) ^(b)—COOH (L-generic-TAT (s)) [SEQ ID NO:7] and/or XXXXRKKRRQ RRRXXXX (L-generic-TAT) [SEQ ID NO: 21], wherein Xor X_(n) ^(b) is as defined above. Furthermore, the number of “X_(n)^(b)” residues in SEQ ID NOs:8 is not limited to the one depicted, andmay vary as described above. Alternatively, the trafficking sequencebeing included in the first domain of the chimeric peptide as usedherein may comprise or consist of a peptide containing e.g. the aminoacid sequence NH₂-GRKKRRQRRR-COOH (L-TAT) [SEQ ID NO: 5].

According to another more preferred embodiment the trafficking sequence(being included in the first domain of the chimeric peptide as usedherein) may comprise a D retro-inverso peptide of the sequences aspresented above, i.e. the D retro-inverso sequence of the generic TATsequence having the sequence NH₂—X_(n) ^(b)-RRRQRRKKR-X_(n) ^(b)—COOH(D-generic-TAT (s)) [SEQ ID NO: 8] and/or XXXXRRRQRRKKRXXXX(D-generic-TAT) [SEQ ID NO: 22]. Also here, X_(n) ^(b) is as definedabove (preferably representing D amino acids). Furthermore, the numberof “X_(n) ^(b)” residues in SEQ ID NOs:8 is not limited to the onedepicted, and may vary as described above. Most preferably, thetrafficking sequence as used herein may comprise the D retro-inversosequence NH₂-RRRQRRKKRG-COOH (D-TAT) [SEQ ID NO: 6].

According to another embodiment the trafficking sequence being includedin the first domain of the chimeric peptide as used herein may compriseor consist of variants of the trafficking sequences as defined above. A“variant of a trafficking sequence” is preferably a sequence derivedfrom a trafficking sequence as defined above, wherein the variantcomprises a modification, for example, addition, (internal) deletion(leading to fragments) and/or substitution of at least one amino acidpresent in the trafficking sequence as defined above. Such (a)modification(s) typically comprise(s) 1 to 20, preferably 1 to 10 andmore preferably 1 to 5 substitutions, additions and/or deletions ofamino acids. Furthermore, the variant preferably exhibits a sequenceidentity with the trafficking sequence as defined above, more preferablywith any of SEQ ID NOs: 5 to 8 or 21-22, of at least about 30%, 50%,70%, 80%, 90%, 95%, 98% or even 99%.

Preferably, such a modification of the trafficking sequence beingincluded in the first domain of the chimeric peptide as used hereinleads to a trafficking sequence with increased or decreased stability.Alternatively, variants of the trafficking sequence can be designed tomodulate intracellular localization of the chimeric peptide as usedherein. When added exogenously, such variants as defined above aretypically designed such that the ability of the trafficking sequence toenter cells is retained (i.e. the uptake of the variant of thetrafficking sequence into the cell is substantially similar to that ofthe native protein used a trafficking sequence). For example, alterationof the basic region thought to be important for nuclear localization(see e.g. Dang and Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauberet al, J. Virol. 63: 1181-1187 (1989); et al., J. Virol. 63: 1-8 (1989))can result in a cytoplasmic location or partially cytoplasmic locationof the trafficking sequence, and therefore, of the JNK inhibitorsequence as component of the chimeric peptide as used herein. Additionalto the above, further modifications may be introduced into the variant,e.g. by linking e.g. cholesterol or other lipid moieties to thetrafficking sequence to produce a trafficking sequence having increasedmembrane solubility. Any of the above disclosed variants of thetrafficking sequences being included in the first domain of the chimericpeptide as used herein can be produced using techniques typically knownto a skilled person (see e.g. Sambrook, J., Fritsch, E. F., Maniatis, T.(1989) Molecular cloning: A laboratory manual. 2nd edition. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.)

As a second domain the chimeric peptide as used herein typicallycomprises an JNK inhibitor sequence, selected from any of the JNKinhibitor sequences as defined above, including variants, fragmentsand/or derivatives of these JNK inhibitor sequences.

Both domains, i.e. the first and the second domain(s), of the chimericpeptide as used herein, may be linked such as to form a functional unit.Any method for linking the first and second domain(s) as generally knownin the art may be applied.

According to one embodiment, the first and the second domain(s) of thechimeric peptide as used herein are preferably linked by a covalentbond. A covalent bond, as defined herein, may be e.g. a peptide bond,which may be obtained by expressing the chimeric peptide as definedabove as a fusion protein. Fusion proteins, as described herein, can beformed and used in ways analogous to or readily adaptable from standardrecombinant DNA techniques, as described below. However, both domainsmay also be linked via side chains or may be linked by a chemical linkermoiety.

The first and/or second domains of the chimeric peptide as used hereinmay occur in one or more copies in said chimeric peptide. If bothdomains are present in a single copy, the first domain may be linkedeither to the N-terminal or the C-terminal end of the second domain. Ifpresent in multiple copies, the first and second domain(s) may bearranged in any possible order. E.g. the first domain can be present inthe chimeric peptide as used herein in a multiple copy number, e.g. intwo, three or more copies, which are preferably arranged in consecutiveorder. Then, the second domain may be present in a single copy occurringat the N- or C-terminus of the sequence comprising the first domain.Alternatively, the second domain may be present in a multiple copynumber, e.g. in two, three or more copies, and the first domain may bepresent in a single copy. According to both alternatives, first andsecond domain(s) can take any place in a consecutive arrangement.Exemplary arrangements are shown in the following: e.g. firstdomain-first domain-first domain-second domain; first domain-firstdomain-second domain-first domain; first domain-second domain-firstdomain-first domain; or e.g. second domain-first domain-firstdomain-first domain. It is well understood for a skilled person thatthese examples are for illustration purposes only and shall not limitthe scope of the invention thereto. Thus, the number of copies and thearrangement may be varied as defined initially.

Preferably, the first and second domain(s) may be directly linked witheach other without any linker. Alternatively, they may be linked witheach other via a linker sequence comprising 1 to 10, preferably 1 to 5amino acids. Amino acids forming the linker sequence are preferablyselected from glycine or proline as amino acid residues. Morepreferably, the first and second domain(s) may be separated by eachother by a hinge of two, three or more proline residues between thefirst and second domain(s).

The chimeric peptide as defined above and as used herein, comprising atleast one first and at least one second domain, may be composed ofL-amino acids, D-amino acids, or a combination of both. Therein, eachdomain (as well as the linkers used) may be composed of L-amino acids,D-amino acids, or a combination of both (e.g. D-TAT and L-IB1(s) orL-TAT and D-IB1(s), etc.). Preferably, the chimeric peptide as usedherein may comprise at least 1 or even 2, preferably at least 3, 4 or 5,more preferably at least 6, 7, 8 or 9 and even more preferably at least10 or more D- and/or L-amino acids, wherein the D- and/or L-amino acidsmay be arranged in the chimeric peptide as used herein in a blockwise, anon-blockwise or in an alternate manner.

According to a specific embodiment the chimeric peptide as used hereincomprises or consists of the L-amino acid chimeric peptides according tothe generic L-TAT-IB peptide NH₂—X_(n) ^(b)-RKKRRQRRR-X_(n) ^(b)—X_(n)^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)—COOH (L-TAT-IB (generic) (s)) [SEQ IDNO: 10], wherein X, X_(n) ^(a) and X_(n) ^(b) are preferably as definedabove. More preferably, the chimeric peptide as used herein comprises orconsists of the L-amino acid chimeric peptideNH₂-GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQD-COOH (L-TAT-IB1 (s)) [SEQ ID NO: 9].Alternatively or additionally, the chimeric peptide as used hereincomprises or consists of the L-amino acid chimeric peptide sequenceGRKKRRQRRR PPDTYRPKRP TTLNLFPQVP RSQDT (L-TAT-IB1) [SEQ ID NO: 23], orXXXXXXXRKK RRQRRRXXXX XXXXRPTTLX LXXXXXXXQD STTX (L-TAT-IB generic) [SEQID NO: 24], wherein X is preferably also as defined above, or thechimeric peptide as used herein comprises or consists of the L-aminoacid chimeric peptide sequence RKKRRQRRRPPRPKRPTTLNLFPQVPRSQD(L-TAT-IB1(s1)) [SEQ ID NO: 27], GRKKRRQRRRX_(n) ^(c)RPKRPTTLNLFPQVPRSQD(L-TAT-IB1(s2)) [SEQ ID NO: 28], or RKKRRQRRRX_(n)^(c)RPKRPTTLNLFPQVPRSQD (L-TAT-IB1(s3)) [SEQ ID NO: 29]. In thiscontext, each X typically represents an amino acid residue as definedabove, more preferably X_(n) ^(c) represents a contiguous stretch ofpeptide residues, each X independently selected from each other fromglycine or proline, e.g. a monotonic glycine stretch or a monotonicproline stretch, wherein n (the number of repetitions of X_(n) ^(c)) istypically 0-5, 5-10, 10-15, 15-20, 20-30 or even more, preferably 0-5 or5-10. X may represent either D or L amino acids.

According to an alternative specific embodiment the chimeric peptide asused herein comprises or consists of D-amino acid chimeric peptides ofthe above disclosed L-amino acid chimeric peptides. Exemplary Dretro-inverso chimeric peptides according to the present invention aree.g. the generic D-TAT-IB peptide NH₂—X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n)^(a)—X_(n) ^(b)-RRRQRRKKR-X_(n) ^(b)—COOH (D-TAT-IB (generic) (s)) [SEQID NO: 12]. Herein, X, X_(n) ^(a) and X_(n) ^(b) are preferably asdefined above (preferably representing D amino acids). More preferably,the chimeric peptide as used herein comprises or consists of D-aminoacid chimeric peptides according to the TAT-IB1 peptideNH₂-DQSRPVQPFLNLTTPRKPRPPRRRQRRKKRG-COOH (D-TAT-IB1(s)) [SEQ ID NO: 11].Alternatively or additionally, the chimeric peptide as used hereincomprises or consists of the D-amino acid chimeric peptide sequenceTDQSRPVQPFLNLTTPRKPRYTDPPRRRQRRKKRG (D-TAT-IB1) [SEQ ID NO: 25], orXT/SDQXXXXXXXLXLTTPRXXXXXXXXRRRQRRKKRXXXXXXX (D-TAT-IB generic) [SEQ IDNO: 26], wherein X is preferably also as defined above, or the chimericpeptide as used herein comprises or consists of the D-amino acidchimeric peptide sequence DQSRPVQPFLNLTTPRKPRPPRRRQRRKKR (D-TAT-IB1(s1))[SEQ ID NO: 30], DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKRG (D-TAT-IB1(s2))[SEQ ID NO: 31], or DQSRPVQPFLNLTTPRKPRX_(n) ^(c)RRRQRRKKR(D-TAT-IB1(s3)) [SEQ ID NO: 32]. X_(n) ^(c) may be as defined above.

The first and second domain(s) of the chimeric peptide as defined abovemay be linked to each other by chemical or biochemical coupling carriedout in any suitable manner known in the art, e.g. by establishing apeptide bond between the first and the second domain(s) e.g. byexpressing the first and second domain(s) as a fusion protein, or e.g.by crosslinking the first and second domain(s) of the chimeric peptideas defined above.

Many known methods suitable for chemical crosslinking of the first andsecond domain(s) of the chimeric peptide as defined above arenon-specific, i.e. they do not direct the point of coupling to anyparticular site on the transport polypeptide or cargo macromolecule. Asa result, use of non-specific crosslinking agents may attack functionalsites or sterically block active sites, rendering the conjugatedproteins biologically inactive. Thus, preferably such crosslinkingmethods are used, which allow a more specific coupling of the first andsecond domain(s).

In this context, one way to increasing coupling specificity is a directchemical coupling to a functional group present only once or a few timesin one or both of the first and second domain(s) to be crosslinked. Forexample, cysteine, which is the only protein amino acid containing athiol group, occurs in many proteins only a few times. Also, forexample, if a polypeptide contains no lysine residues, a crosslinkingreagent specific for primary amines will be selective for the aminoterminus of that polypeptide. Successful utilization of this approach toincrease coupling specificity requires that the polypeptide have thesuitably rare and reactive residues in areas of the molecule that may bealtered without loss of the molecule's biological activity. Cysteineresidues may be replaced when they occur in parts of a polypeptidesequence where their participation in a crosslinking reaction wouldotherwise likely interfere with biological activity. When a cysteineresidue is replaced, it is typically desirable to minimize resultingchanges in polypeptide folding. Changes in polypeptide folding areminimized when the replacement is chemically and sterically similar tocysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for crosslinkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, wherein thepolypeptide of interest is produced by chemical synthesis or viaexpression of recombinant DNA.

Coupling of the first and second domain(s) of the chimeric peptide asdefined above and used herein can also be accomplished via a coupling orconjugating agent. There are several intermolecular crosslinkingreagents which can be utilized (see for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43). Amongthese reagents are, for example, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene)bismaleimide(both of which are highly specific for sulfhydryl groups and formirreversible linkages); N,N′-ethylene-bis-(iodoacetamide) or other suchreagent having 6 to 11 carbon methylene bridges (which are relativelyspecific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene(which forms irreversible linkages with amino and tyrosine groups).Other crosslinking reagents useful for this purpose include:p,p′-difluoro-m, m′-dinitrodiphenylsulfone which forms irreversiblecrosslinkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4 disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor di isothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Crosslinking reagents used for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above may behomobifunctional, i.e. having two functional groups that undergo thesame reaction. A preferred homobifunctional crosslinking reagent isbismaleimidohexane (“BMH”). BMH contains two maleimide functionalgroups, which react specifically with sulfhydryl-containing compoundsunder mild conditions (pH 6.5-7.7). The two maleimide groups areconnected by a hydrocarbon chain. Therefore, BMH is useful forirreversible crosslinking of polypeptides that contain cysteineresidues.

Crosslinking reagents used for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above may also beheterobifunctional. Heterobifunctional crosslinking agents have twodifferent functional groups, for example an amine-reactive group and athiol-reactive group, that will crosslink two proteins having freeamines and thiols, respectively. Examples of heterobifunctionalcrosslinking agents are succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccin imide ester (“MBS”), and succinimide4-(p-maleimidophenyl)butyrate (“SMPB”), an extended chain analog of MBS.The succinimidyl group of these crosslinkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Crosslinking reagents suitable for crosslinking the first and seconddomain(s) of the chimeric peptide as defined above often have lowsolubility in water. A hydrophilic moiety, such as a sulfonate group,may thus be added to the crosslinking reagent to improve its watersolubility. In this respect, Sulfo-MBS and Sulfo-SMCC are examples ofcrosslinking reagents modified for water solubility, which may be usedaccording to the present invention.

Likewise, many crosslinking reagents yield a conjugate that isessentially non-cleavable under cellular conditions. However, somecrosslinking reagents particularly suitable for crosslinking the firstand second domain(s) of the chimeric peptide as defined above contain acovalent bond, such as a disulfide, that is cleavable under cellularconditions. For example, Traut's reagent,dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavablecrosslinkers. The use of a cleavable crosslinking reagent permits thecargo moiety to separate from the transport polypeptide after deliveryinto the target cell. Direct disulfide linkage may also be useful.

Numerous crosslinking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincrosslinking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSSLINKING, CRC Press (1991).

Chemical crosslinking of the first and second domain(s) of the chimericpeptide as defined above may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the crosslinking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

Furthermore, variants, fragments or derivatives of one of the abovedisclosed chimeric peptides may be used herein. With regard to fragmentsand variants it is generally referred to the definition given above forJNK inhibitor sequences.

Particularly, in the context of the present invention, a “variant of achimeric peptide” is preferably a sequence derived from any of thesequences according to SEQ ID NOs: 9 to 12 and 23 to 32, wherein thechimeric variant comprises amino acid alterations of the chimericpeptides according to SEQ ID NOs: 9 to 12 and 23 to 32 as used herein.Such alterations typically comprise 1 to 20, preferably 1 to 10 and morepreferably 1 to 5 substitutions, additions and/or deletions (leading tofragments) of amino acids according to SEQ ID NOs: 9 to 12 and 23 to 32,wherein the altered chimeric peptide as used herein exhibits a sequenceidentity with any of the sequences according to SEQ ID NOs: 9-12 and 23to 32 of at least about 30%, 50%, 70%, 80%, or 95%, 98%, or even 99%.Preferably, these variants retain the biological activity of the firstand the second domain as contained in the chimeric peptide as usedherein, i.e. the trafficking activity of the first domain as disclosedabove and the activity of the second domain for binding JNK and/orinhibiting the activation of at least one JNK activated transcriptionfactor.

Accordingly, the chimeric peptide as used herein also comprisesfragments of the afore disclosed chimeric peptides, particularly of thechimeric peptide sequences according to any of SEQ ID NOs: 9 to 12 and23 to 32. Thus, in the context of the present invention, a “fragment ofthe chimeric peptide” is preferably a sequence derived any of thesequences according to SEQ ID NOs: 9 to 12 and 23 to 32, wherein thefragment comprises at least 4 contiguous amino acids of any of SEQ IDNOs: 9 to 12 and 23 to 32. This fragment preferably comprises a lengthwhich is sufficient to allow specific recognition of an epitope from anyof these sequences and to transport the sequence into the cells, thenucleus or a further preferred location. Even more preferably, thefragment comprises 4 to 18, 4 to 15, or most preferably 4 to 10contiguous amino acids of any of SEQ ID NOs: 9 to 12 and 23 to 32.Fragments of the chimeric peptide as used herein further may be definedas a sequence sharing a sequence identity with any of the sequencesaccording to any of SEQ ID NOs: 99 to 12 and 23 to 32 of at least about30%, 50%, 70%, 80%, or 95%, 98%, or even 99%.

Finally, the chimeric peptide as used herein also comprises derivativesof the afore disclosed chimeric peptides, particularly of the chimericpeptide sequences according to any of SEQ ID NOs: 9 to 12 and 23 to 32.

The present invention additionally refers to the use of nucleic acidsequences encoding JNK inhibitor sequences as defined above, chimericpeptides or their fragments, variants or derivatives, all as definedabove, for the preparation of a pharmaceutical composition for treatingnon-chronic or chronic inflammatory digestive diseases in a subject asdefined herein. A preferable suitable nucleic acid encoding an JNKinhibitor sequence as used herein is typically chosen from human IB1nucleic acid (GenBank Accession No. (AF074091), rat IB1 nucleic acid(GenBank Accession No. AF 108959), or human IB2 (GenBank Accession NoAF218778) or from any nucleic acid sequence encoding any of thesequences as defined above, i.e. any sequence according to SEQ ID NO:1-26.

Nucleic acids encoding the JNK inhibitor sequences as used herein orchimeric peptides as used herein may be obtained by any method known inthe art (e.g. by PCR amplification using synthetic primers hybridizableto the 3′- and 5′-termini of the sequence and/or by cloning from a cDNAor genomic library using an oligonucleotide sequence specific for thegiven gene sequence).

Additionally, nucleic acid sequences are disclosed herein as well, whichhybridize under stringent conditions with the appropriate strand codingfor a (native) JNK inhibitor sequence or chimeric peptide as definedabove. Preferably, such nucleic acid sequences comprise at least 6(contiguous) nucleic acids, which have a length sufficient to allow forspecific hybridization. More preferably, such nucleic acid sequencescomprise 6 to 38, even more preferably 6 to 30, and most preferably 6 to20 or 6 to 10 (contiguous) nucleic acids.

“Stringent conditions” are sequence dependent and will be differentunder different circumstances. Generally, stringent conditions can beselected to be about 5° C. lower than the thermal melting point (TM) forthe specific sequence at a defined ionic strength and pH. The TM is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

“High stringency conditions” may comprise the following, e.g. Step 1:Filters containing DNA are pretreated for 8 hours to overnight at 65° C.in buffer composed of 6*SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Step 2: Filters are hybridized for 48 hours at 65° C. in the aboveprehybridization mixture to which is added 100 mg/ml denatured salmonsperm DNA and 5−20*10⁶ cpm of ³²P-labeled probe. Step 3: Filters arewashed for 1 hour at 37° C. in a solution containing 2*SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1*SSC at50° C. for 45 minutes. Step 4: Filters are autoradiographed. Otherconditions of high stringency that may be used are well known in the art(see e.g. Ausubel et al, (eds.), 1993, Current Protocols in MolecularBiology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer andExpression, a Laboratory Manual, Stockton Press, NY).

“Moderate stringency conditions” can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6*SSC, 5*Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20hours at 55° C. in the same solution with 5−20*10⁶ cpm ³²P-labeled probeadded. Step 3: Filters are washed at 37° C. for 1 hour in a solutioncontaining 2*SSC, 0.1% SDS, then washed twice for 30 minutes at 60° C.in a solution containing 1*SSC and 0.1% SDS. Step 4: Filters are blotteddry and exposed for autoradiography. Other conditions of moderatestringency that may be used are well-known in the art (see e.g. Ausubelet al., (eds.), 1993, Current Protocols in Molecular Biology, John Wileyand Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, aLaboratory Manual, Stockton Press, NY).

Finally, “low stringency conditions” can include: Step 1: Filterscontaining DNA are pretreated for 6 hours at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA.Step 2: Filters are hybridized for 18-20 hours at 40° C. in the samesolution with the addition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5−20×106 cpm³²P-labeled probe. Step 3: Filters are washed for 1.5 hours at 55 C in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Step 4: Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and reexposed to film. Other conditions of lowstringency that may be used are well known in the art (e.g. as employedfor cross-species hybridizations). See e.g. Ausubel et al., (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, NY;and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY.

The nucleic acid sequences as defined above according to the presentinvention can be used to express peptides, i.e. an JNK inhibitorsequence as used herein or an chimeric peptide as used herein foranalysis, characterization or therapeutic use; as markers for tissues inwhich the corresponding peptides (as used herein) are preferentiallyexpressed (either constitutively or at a particular stage of tissuedifferentiation or development or in disease states). Other uses forthese nucleic acids include, e.g. molecular weight markers in gelelectrophoresis-based analysis of nucleic acids.

According to a further embodiment of the present invention, expressionvectors may be used for the above purposes for recombinant expression ofone or more JNK inhibitor sequences and/or chimeric peptides as definedabove. The term “expression vector” is used herein to designate eithercircular or linear DNA or RNA, which is either double-stranded orsingle-stranded. It further comprises at least one nucleic acid asdefined above to be transferred into a host cell or into a unicellularor multicellular host organism. The expression vector as used hereinpreferably comprises a nucleic acid as defined above encoding the JNKinhibitor sequence as used herein or a fragment or a variant thereof, orthe chimeric peptide as used herein, or a fragment or a variant thereof.Additionally, an expression vector according to the present inventionpreferably comprises appropriate elements for supporting expressionincluding various regulatory elements, such as enhancers/promoters fromviral, bacterial, plant, mammalian, and other eukaryotic sources thatdrive expression of the inserted polynucleotide in host cells, such asinsulators, boundary elements, LCRs (e.g. described by Blackwood andKadonaga (1998), Science 281, 61-63) or matrix/scaffold attachmentregions (e.g. described by Li, Harju and Peterson, (1999), Trends Genet.15, 403-408). In some embodiments, the regulatory elements areheterologous (i.e. not the native gene promoter). Alternately, thenecessary transcriptional and translational signals may also be suppliedby the native promoter for the genes and/or their flanking regions.

The term “promoter” as used herein refers to a region of DNA thatfunctions to control the transcription of one or more nucleic acidsequences as defined above, and that is structurally identified by thepresence of a binding site for DNA-dependent RNA-polymerase and of otherDNA sequences, which interact to regulate promoter function. Afunctional expression promoting fragment of a promoter is a shortened ortruncated promoter sequence retaining the activity as a promoter.Promoter activity may be measured by any assay known in the art (seee.g. Wood, de Wet, Dewji, and DeLuca, (1984), Biochem Biophys. Res.Commun. 124, 592-596; Seliger and McElroy, (1960), Arch. Biochem.Biophys. 88, 136-141) or commercially available from Promega®).

An “enhancer region” to be used in the expression vector as definedherein, typically refers to a region of DNA that functions to increasethe transcription of one or more genes. More specifically, the term“enhancer”, as used herein, is a DNA regulatory element that enhances,augments, improves, or ameliorates expression of a gene irrespective ofits location and orientation vis-à-vis the gene to be expressed, and maybe enhancing, augmenting, improving, or ameliorating expression of morethan one promoter.

The promoter/enhancer sequences to be used in the expression vector asdefined herein, may utilize plant, animal, insect, or fungus regulatorysequences. For example, promoter/enhancer elements can be used fromyeast and other fungi (e.g. the GAL4 promoter, the alcohol dehydrogenasepromoter, the phosphoglycerol kinase promoter, the alkaline phosphatasepromoter). Alternatively, or in addition, they may include animaltranscriptional control regions, e.g. (i) the insulin gene controlregion active within pancreatic beta-cells (see e.g. Hanahan, et al,1985. Nature 315: 115-122); (ii) the immunoglobulin gene control regionactive within lymphoid cells (see e.g. Grosschedl, et al, 1984, Cell 38:647-658); (iii) the albumin gene control region active within liver (seee.g. Pinckert, et al, 1987. Genes and Dev 1: 268-276; (iv) the myelinbasic protein gene control region active within brain oligodendrocytecells (see e.g. Readhead, et al, 1987, Cell 48: 703-712); and (v) thegonadotropin-releasing hormone gene control region active within thehypothalamus (see e.g. Mason, et al, 1986, Science 234: 1372-1378), andthe like.

Additionally, the expression vector as defined herein may comprise anamplification marker. This amplification marker may be selected from thegroup consisting of, e.g. adenosine deaminase (ADA), dihydrofolatereductase (DHFR), multiple drug resistance gene (MDR), ornithinedecarboxylase (ODC) and N-(phosphonacetyl)-L-aspartate resistance (CAD).

Exemplary expression vectors or their derivatives suitable for thepresent invention particularly include, e.g. human or animal viruses(e.g. vaccinia virus or adenovirus); insect viruses (e.g. baculovirus);yeast vectors; bacteriophage vectors (e.g. lambda phage); plasmidvectors and cosmid vectors.

The present invention additionally may utilize a variety of host-vectorsystems, which are capable of expressing the peptide coding sequence(s)of nucleic acids as defined above. These include, but are not limitedto: (i) mammalian cell systems that are infected with vaccinia virus,adenovirus, and the like; (ii) insect cell systems infected withbaculovirus and the like; (iii) yeast containing yeast vectors or (iv)bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. Depending upon the host-vector system utilized, any one of a numberof suitable transcription and translation elements may be used.

Preferably, a host cell strain, suitable for such a host-vector system,may be selected that modulates the expression of inserted sequences ofinterest, or modifies or processes expressed peptides encoded by thesequences in the specific manner desired. In addition, expression fromcertain promoters may be enhanced in the presence of certain inducers ina selected host strain; thus facilitating control of the expression of agenetically-engineered peptide. Moreover, different host cells possesscharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g. glycosylation,phosphorylation, and the like) of expressed peptides. Appropriate celllines or host systems may thus be chosen to ensure the desiredmodification and processing of the foreign peptide is achieved. Forexample, peptide expression within a bacterial system can be used toproduce an non-glycosylated core peptide; whereas expression withinmammalian cells ensures “native” glycosylation of a heterologouspeptide.

The present invention further provides the use of antibodies directedagainst the JNK inhibitor sequences and/or chimeric peptides asdescribed above, for preparing a pharmaceutical composition for thetreatment of non-chronic or chronic inflammatory digestive diseases asdefined herein. Furthermore, efficient means for production ofantibodies specific for JNK inhibitor sequences according to the presentinvention, or for chimeric peptides containing such an inhibitorsequence, are described and may be utilized for this purpose.

According to the invention, JNK inhibitor sequences and/or chimericpeptides as defined herein, as well as, fragments, variants orderivatives thereof, may be utilized as immunogens to generateantibodies that immunospecifically bind these peptide components. Suchantibodies include, e.g. polyclonal, monoclonal, chimeric, single chain,Fab fragments and a Fab expression library. In a specific embodiment thepresent invention provides antibodies to chimeric peptides or to JNKinhibitor sequences as defined above. Various procedures known withinthe art may be used for the production of these antibodies.

By way of example, various host animals may be immunized for productionof polyclonal antibodies by injection with any chimeric peptide or JNKinhibitor sequence as defined above. Various adjuvants may be usedthereby to increase the immunological response which include, but arenot limited to, Freund's (complete and incomplete) adjuvant, mineralgels (e.g. aluminum hydroxide), surface active substances (e.g.lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, etc.), CpG, polymers, Pluronics, and human adjuvants suchas Bacille Calmette-Guerin and Corynebacterium parvum.

For preparation of monoclonal antibodies directed towards a chimericpeptide or a JNK inhibitor sequence as defined above, any technique maybe utilized that provides for the production of antibody molecules bycontinuous cell line culture. Such techniques include, but are notlimited to, the hybridoma technique (see Kohler and Milstein, 1975.Nature 256: 495-497); the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al, 1983, Immunol Today 4: 72) and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, et1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Human monoclonal antibodies may be utilized in the practiceof the present invention and may be produced by the use of humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et a/., 1985. In: Monoclonal Antibodies and CancerTherapy (Alan R. Liss, Inc., pp. 77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to the INK inhibitor sequencesand/or chimeric peptides (see e.g. U.S. Pat. No. 4,946,778) as definedherein. In addition, methods can be adapted for the construction of Fabexpression libraries (see e.g. Huse et al., 1989. Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fabfragments with the desired specificity for these JNK inhibitor sequencesand/or chimeric peptides. Non-human antibodies can be “humanized” bytechniques well known in the art (see e.g. U.S. Pat. No. 5,225,539).Antibody fragments that contain the idiotypes to a JNK inhibitorsequences and/or chimeric peptide as defined herein may be produced bytechniques known in the art including, e.g. (i) a F(ab′)₂ fragmentproduced by pepsin digestion of an antibody molecule; (ii) a Fabfragment generated by reducing the disulfide bridges of an F(ab′)₂fragment; (iii) a Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) Fvfragments.

In one embodiment of this invention, methods, that may be utilized forthe screening of antibodies and which possess the desired specificityinclude, but are not limited to, enzyme-linked immunosorbent assay(ELISA) and other immunologically-mediated techniques known within theart. In a specific embodiment, selection of antibodies that are specificto a particular epitope of an JNK inhibitor sequence and/or an chimericpeptide as defined herein (e.g. a fragment thereof typically comprisinga length of from 5 to 20, preferably 8 to 18 and most preferably 8 to 11amino acids) is facilitated by generation of hybridomas that bind to thefragment of an JNK inhibitor sequence and/or an chimeric peptide, asdefined herein, possessing such an epitope. These antibodies that arespecific for an epitope as defined above are also provided herein.

The antibodies as defined herein may be used in methods known within theart referring to the localization and/or quantification of an JNKinhibitor sequence (and/or correspondingly to a chimeric peptide asdefined above), e.g. for use in measuring levels of the peptide withinappropriate physiological samples, for use in diagnostic methods, or foruse in imaging the peptide, and the like.

The JNK inhibitor sequences, chimeric peptides, nucleic acids, vectors,host cells and/or antibodies as defined according to the invention canbe formulated in a pharmaceutical composition, which may be applied inthe prevention or treatment of any of the diseases as defined herein,particularly in the prevention or treatment of non-chronic or chronicinflammatory digestive diseases as defined herein. Typically, such apharmaceutical composition used according to the present inventionincludes as an active component, e.g.: (i) any one or more of the JNKinhibitor sequences and/or chimeric peptides as defined above, and/orvariants, fragments or derivatives thereof, particularly JNK inhibitorsequences according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 and/or chimeric peptides according to any of sequences ofSEQ ID NOs: 9 to 12 and 23 to 32, and/or JNK inhibitor sequencesaccording to any of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and33-100 comprising a trafficking sequence according to any of SEQ ID NOs:5 to 8 and 21 to 22, or variants or fragments thereof within the abovedefinitions; and/or (ii) nucleic acids encoding an JNK inhibitorsequence and/or an chimeric peptide as defined above and/or variants orfragments thereof, and/or (iii) cells comprising any one or more of theJNK inhibitor sequences and/or chimeric peptides, and/or variants,fragments or derivatives thereof, as defined above and/or (iv) cellstransfected with a vector and/or nucleic acids encoding an JNK inhibitorsequence and/or an chimeric peptide as defined above and/or variants orfragments thereof.

According to a preferred embodiment, such a pharmaceutical compositionas used according to the present invention typically comprises a safeand effective amount of a component as defined above, preferably of atleast one JNK inhibitor sequence according to any of sequences of SEQ IDNOs: 1 to 4 and 13 to 20 and 33-100 and/or at least one chimeric peptideaccording to any of sequences of SEQ ID NOs: 9 to 12 and 23 to 32,and/or at least one JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising a traffickingsequence according to any of SEQ ID NOs: 5-8 and 21 to 22, or variantsor fragments thereof within the above definitions, or at least onenucleic acids encoding same, or at least one vector, host cell orantibody as defined above.

The inventors of the present invention additionally found, that theJNK-inhibitor sequence and the chimeric peptide, respectively, asdefined herein, exhibit a particular well uptake rate into cellsinvolved in the diseases of the present invention. Therefore, the amountof a JNK-inhibitor sequence and chimeric peptide, respectively, in thepharmaceutical composition to be administered to a subject, may—withoutbeing limited thereto—have a very low dose. Thus, the dose may be muchlower than for peptide drugs known in the art, such as DTS-108 (FlorenceMeyer-Losic et al., Clin Cancer Res., 2008, 2145-53). This has severalpositive aspects, for example a reduction of potential side reactionsand a reduction in costs.

Preferably, the dose (per kg bodyweight) is in the range of up to 10mmol/kg, preferably up to 1 mmol/kg, more preferably up to 100 μmol/kg,even more preferably up to 10 μmol/kg, even more preferably up to 1μmol/kg, even more preferably up to 100 nmol/kg, most preferably up to50 nmol/kg.

Thus, the dose range may preferably be from about 1 pmol/kg to about 1mmol/kg, from about 10 pmol/kg to about 0.1 mmol/kg, from about 10pmol/kg to about 0.01 mmol/kg, from about 50 pmol/kg to about 1 pmol/kg,from about 100 pmol/kg to about 500 nmol/kg, from about 200 pmol/kg toabout 300 nmol/kg, from about 300 pmol/kg to about 100 nmol/kg, fromabout 500 pmol/kg to about 50 nmol/kg, from about 750 pmol/kg to about30 nmol/kg, from about 250 pmol/kg to about 5 nmol/kg, from about 1nmol/kg to about 10 nmol/kg, or a combination of any two of said values.

In this context, prescription of treatment, e.g. decisions on dosageetc. when using the above pharmaceutical composition is typically withinthe responsibility of general practitioners and other medical doctors,and typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980. Accordingly,a “safe and effective amount” as defined above for components of thepharmaceutical compositions as used according to the present inventionmeans an amount of each or all of these components, that is sufficientto significantly induce a positive modification of a non-chronic orchronic inflammatory digestive diseases as defined herein. At the sametime, however, a “safe and effective amount” is small enough to avoidserious side-effects, that is to say to permit a sensible relationshipbetween advantage and risk. The determination of these limits typicallylies within the scope of sensible medical judgment. A “safe andeffective amount” of such a component will vary in connection with theparticular condition to be treated and also with the age and physicalcondition of the patient to be treated, the severity of the condition,the duration of the treatment, the nature of the accompanying therapy,of the particular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the accompanying doctor.The pharmaceutical compositions according to the invention can be usedaccording to the invention for human and also for veterinary medicalpurposes.

The pharmaceutical composition as used according to the presentinvention may furthermore comprise, in addition to one of thesesubstances, a (compatible) pharmaceutically acceptable carrier,excipient, buffer, stabilizer or other materials well known to thoseskilled in the art.

In this context, the expression “(compatible) pharmaceuticallyacceptable carrier” preferably includes the liquid or non-liquid basisof the composition. The term “compatible” means that the constituents ofthe pharmaceutical composition as used herein are capable of being mixedwith the pharmaceutically active component as defined above and with oneanother component in such a manner that no interaction occurs whichwould substantially reduce the pharmaceutical effectiveness of thecomposition under usual use conditions. Pharmaceutically acceptablecarriers must, of course, have sufficiently high purity and sufficientlylow toxicity to make them suitable for administration to a person to betreated.

If the pharmaceutical composition as used herein is provided in liquidform, the pharmaceutically acceptable carrier will typically compriseone or more (compatible) pharmaceutically acceptable liquid carriers.The composition may comprise as (compatible) pharmaceutically acceptableliquid carriers e.g. pyrogen-free water; isotonic saline or buffered(aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions,vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid, etc. Particularly for injection ofthe pharmaceutical composition as used herein, a buffer, preferably anaqueous buffer, may be used.

If the pharmaceutical composition as used herein is provided in solidform, the pharmaceutically acceptable carrier will typically compriseone or more (compatible) pharmaceutically acceptable solid carriers. Thecomposition may comprise as (compatible) pharmaceutically acceptablesolid carriers e.g. one or more compatible solid or liquid fillers ordiluents or encapsulating compounds may be used as well, which aresuitable for administration to a person. Some examples of such(compatible) pharmaceutically acceptable solid carriers are e.g. sugars,such as, for example, lactose, glucose and sucrose; starches, such as,for example, corn starch or potato starch; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin;tallow; solid glidants, such as, for example, stearic acid, magnesiumstearate; calcium sulphate, etc.

The precise nature of the (compatible) pharmaceutically acceptablecarrier or other material may depend on the route of administration. Thechoice of a (compatible) pharmaceutically acceptable carrier may thus bedetermined in principle by the manner in which the pharmaceuticalcomposition as used according to the invention is administered. Thepharmaceutical composition as used according to the invention can beadministered, for example, systemically. Routes for administrationinclude, for example, parenteral routes (e.g. via injection), such asintravenous, intramuscular, subcutaneous, intradermal, or transdermalroutes, etc., enteral routes, such as oral, or rectal routes, etc.,topical routes, such as nasal, or intranasal routes, etc., or otherroutes, such as epidermal routes or patch delivery.

The suitable amount of the pharmaceutical composition to be used can bedetermined by routine experiments with animal models. Such modelsinclude, without implying any limitation, rabbit, sheep, mouse, rat, dogand non-human primate models. Preferred unit dose forms for injectioninclude sterile solutions of water, physiological saline or mixturesthereof. The pH of such solutions should be adjusted to about 7.4.Suitable carriers for injection include hydrogels, devices forcontrolled or delayed release, polylactic acid and collagen matrices.Suitable pharmaceutically acceptable carriers for topical applicationinclude those, which are suitable for use in lotions, creams, gels andthe like. If the compound is to be administered perorally, tablets,capsules and the like are the preferred unit dose form. Thepharmaceutically acceptable carriers for the preparation of unit doseforms, which can be used for oral administration are well known in theprior art. The choice thereof will depend on secondary considerationssuch as taste, costs and storability, which are not critical for thepurposes of the present invention, and can be made without difficulty bya person skilled in the art.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carrier asdefined above, such as gelatin, and optionally an adjuvant. Liquidpharmaceutical compositions for oral administration generally mayinclude a liquid carrier as defined above, such as water, petroleum,animal or vegetable oils, mineral oil or synthetic oil. Physiologicalsaline solution, dextrose or other saccharide solution or glycols suchas ethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required.Whether it is a polypeptide, peptide, or nucleic acid molecule, otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount or a “therapeutically effectiveamount” (as the case may be), this being sufficient to show benefit tothe individual. The actual amount administered, and rate and time-courseof administration, will depend on the nature and severity of what isbeing treated.

Prevention and/or treatment of a disease as defined herein typicallyincludes administration of a pharmaceutical composition as definedabove. The term “modulate” includes the suppression of expression of JNKwhen it is over-expressed in any of the above diseases. It alsoincludes, without being limited thereto, suppression of phosphorylationof c-jun, ATF2 or NFAT4 in any of the above diseases, for example, byusing at least one JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or at least onechimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, and/or at least one JNK inhibitor sequence according toany of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100comprising a trafficking sequence according to any of SEQ ID NOs: 5 to 8and 21 to 22, or variants or fragments thereof within the abovedefinitions, as a competitive inhibitor of the natural c-jun, ATF2 andNFAT4 binding site in a cell. The term “modulate” also includessuppression of hetero- and homomeric complexes of transcription factorsmade up of, without being limited thereto, c-jun, ATF2, or NFAT4 andtheir related partners, such as for example the AP-1 complex that ismade up of c-jun, AFT2 and c-fos. When a non-chronic or chronicinflammatory digestive disease is associated with JNK overexpression,such suppressive JNK inhibitor sequences can be introduced to a cell. Insome instances, “modulate” may then include the increase of JNKexpression, for example by use of an IB peptide-specific antibody thatblocks the binding of an IB-peptide to JNK, thus preventing JNKinhibition by the IB-related peptide.

Prevention and/or treatment of a subject with the pharmaceuticalcomposition as disclosed above may be typically accomplished byadministering (in vivo) an (“therapeutically effective”) amount of saidpharmaceutical composition to a subject, wherein the subject may be e.g.any mammal, e.g. a human, a primate, mouse, rat, dog, cat, cow, horse orpig. The term “therapeutically effective” means that the activecomponent of the pharmaceutical composition is of sufficient quantity toameliorate the non-chronic or chronic inflammatory digestive disease.

Accordingly, peptides as defined above, e.g. at least one JNK inhibitorsequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 and/or at least one chimeric peptide according to any ofsequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or at least one JNKinhibitor sequence according to any of sequences of SEQ ID NOs: 1 to 4and 13 to 20 and 33-100 comprising a trafficking sequence according toany of SEQ ID NOs: 5 to 8 and 21 to 22, or variants or fragments thereofwithin the above definitions, may be utilized in a specific embodimentof the present invention to treat non-chronic or chronic inflammatorydigestive diseases as defined above by modulating activated JNKsignaling pathways.

Peptides as defined above and as contained in the inventivepharmaceutical composition may be also encoded by nucleic acids. This isparticularly advantageous, if the above peptides are administered forthe purpose of gene therapy. In this context, gene therapy refers totherapy that is performed by administration of a specific nucleic acidas defined above to a subject, e.g. by way of a pharmaceuticalcomposition as defined above, wherein the nucleic acid(s) exclusivelycomprise(s) L-amino acids. In this embodiment of the present invention,the nucleic acid produces its encoded peptide(s), which then serve(s) toexert a therapeutic effect by modulating function of the disease ordisorder. Any of the methods relating to gene therapy available withinthe art may be used in the practice of the present invention (see e.g.Goldspiel, et al, 1993. Clin Pharm 12: 488-505).

In a preferred embodiment, the nucleic acid as defined above and as usedfor gene therapy is part of an expression vector encoding and expressingany one or more of the 1B-related peptides as defined above within asuitable host, i.e. an JNK inhibitor sequence according to any ofsequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 and/or achimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, and/or an JNK inhibitor sequence according to any ofsequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising atrafficking sequence according to any of SEQ ID NOs: 5 to 8 and 21 to22, or variants or fragments thereof within the above definitions. In aspecific embodiment, such an expression vector possesses a promoter thatis operably-linked to coding region(s) of a JNK inhibitor sequence. Thepromoter may be defined as above, e.g. inducible or constitutive, and,optionally, tissue-specific.

In another specific embodiment, a nucleic acid molecule as defined aboveis used for gene therapy, in which the coding sequences of the nucleicacid molecule (and any other desired sequences thereof) as defined aboveare flanked by regions that promote homologous recombination at adesired site within the genome, thus providing for intra-chromosomalexpression of these nucleic acids (see e.g. Koller and Smithies, 1989.Proc Natl Acad Sci USA 86: 8932-8935).

Delivery of the nucleic acid as defined above according to the inventioninto a patient for the purpose of gene therapy, particular in thecontext of the above mentioned non-chronic or chronic inflammatorydigestive diseases as defined above may be either direct (i.e. thepatient is directly exposed to the nucleic acid or nucleicacid-containing vector) or indirect (i.e. cells are first transformedwith the nucleic acid in vitro, then transplanted into the patient).These two approaches are known, respectively, as in vivo or ex vivo genetherapy. In a specific embodiment of the present invention, a nucleicacid is directly administered in vivo, where it is expressed to producethe encoded product. This may be accomplished by any of numerous methodsknown in the art including, e.g. constructing the nucleic acid as partof an appropriate nucleic acid expression vector and administering thesame in a manner such that it becomes intracellular (e.g. by infectionusing a defective or attenuated retroviral or other viral vector; seeU.S. Pat. No. 4,980,286); directly injecting naked DNA; usingmicroparticle bombardment (e.g. a “GeneGun”; Biolistic, DuPont); coatingthe nucleic acids with lipids; using associated cell-surfacereceptors/transfecting agents; encapsulating in liposomes,microparticles, or microcapsules; administering it in linkage to apeptide that is known to enter the nucleus; or by administering it inlinkage to a ligand predisposed to receptor-mediated endocytosis (seee.g. Wu and Wu, 1987.) Biol Chem 262: 4429-4432), which can be used to“target” cell types that specifically express the receptors of interest,etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a nucleic acid as defined above intocells in in vitro tissue culture by such methods as electroporation,lipofection, calcium phosphate-mediated transfection, viral infection,or the like. Generally, the method of transfer includes the concomitanttransfer of a selectable marker to the cells. The cells are then placedunder selection pressure (e.g. antibiotic resistance) so as tofacilitate the isolation of those cells that have taken up, and areexpressing, the transferred gene. Those cells are then delivered to apatient. In a specific embodiment, prior to the in vivo administrationof the resulting recombinant cell, the nucleic acid is introduced into acell by any method known within the art including e.g. transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences of interest, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, and similar methods that ensure that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted by the transfer. See e.g. Loeffler and Behr, 1993. MethEnzymol 217: 599-618. The chosen technique should provide for the stabletransfer of the nucleic acid to the cell, such that the nucleic acid isexpressible by the cell. Preferably, the transferred nucleic acid isheritable and expressible by the cell progeny.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, e.g. injection of epithelial cells (e.g.subcutaneously), application of recombinant skin cells as a skin graftonto the patient, and intravenous injection of recombinant blood cells(e.g. hematopoietic stem or progenitor cells). The total amount of cellsthat are envisioned for use depend upon the desired effect, patientstate, and the like, and may be determined by one skilled within theart. Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g. asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

Alternatively and/or additionally, for treating diseases as mentionedherein targeting therapies may be used to deliver the JNK inhibitorsequences, chimeric peptides, and/or nucleic acids as defined above morespecifically to certain types of cell, by the use of targeting systemssuch as (a targeting) antibody or cell specific ligands. Antibodies usedfor targeting are typically specific for cell surface proteins of cellsassociated with any of the diseases as defined below. By way of example,these antibodies may be directed to cell surface antibodies such as e.g.B cell-associated surface proteins such as MHC class II DR protein, CD18(LFA-1 beta chain), CD45RO, CD40 or Bgp95, or cell surface proteinsselected from e.g. CD2, CD2, CD4, CD5, CD7, CD8, CD9, CD10, CD13, CD16,CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD38, CD39,CD4, CD43, CD45, CD52, CD56, CD68, CD71, CD138, etc. Targetingconstructs may be typically prepared by covalently binding the JNKinhibitor sequences, chimeric peptides, and nucleic acids as definedherein according to the invention to an antibody specific for a cellsurface protein or by binding to a cell specific ligand. Proteins maye.g. be bound to such an antibody or may be attached thereto by apeptide bond or by chemical coupling, crosslinking, etc. The targetingtherapy may then be carried out by administering the targeting constructin a pharmaceutically efficient amount to a patient by any of theadministration routes as defined below, e.g. intraperitoneal, nasal,intravenous, oral and patch delivery routes. Preferably, the JNKinhibitor sequences, chimeric peptides, or nucleic acids as definedherein according to the invention, being attached to the targetingantibodies or cell specific ligands as defined above, may be released invitro or in vivo, e.g. by hydrolysis of the covalent bond, by peptidasesor by any other suitable method. Alternatively, if the JNK inhibitorsequences, chimeric peptides, or nucleic acids as defined hereinaccording to the invention are attached to a small cell specific ligand,release of the ligand may not be carried out. If present at the cellsurface, the chimeric peptides may enter the cell upon the activity ofits trafficking sequence. Targeting may be desirable for a variety ofreasons; for example if the JNK inhibitor sequences, chimeric peptides,and nucleic acids as defined herein according to the invention areunacceptably toxic or if it would otherwise require a too high dosage.

Instead of administering the JNK inhibitor sequences and/or chimericpeptides as defined herein according to the invention directly, theycould be produced in the target cells by expression from an encodinggene introduced into the cells, e.g. from a viral vector to beadministered. The viral vector typically encodes the JNK inhibitorsequences and/or chimeric peptides as defined herein according to theinvention. The vector could be targeted to the specific cells to betreated. Moreover, the vector could contain regulatory elements, whichare switched on more or less selectively by the target cells upondefined regulation. This technique represents a variant of the VDEPTtechnique (virus-directed enzyme prodrug therapy), which utilizes matureproteins instead of their precursor forms.

Alternatively, the JNK inhibitor sequences and/or chimeric peptides asdefined herein could be administered in a precursor form by use of anantibody or a virus. These JNK inhibitor sequences and/or chimericpeptides may then be converted into the active form by an activatingagent produced in, or targeted to, the cells to be treated. This type ofapproach is sometimes known as ADEPT (antibody-directed enzyme prodrugtherapy) or VDEPT (virus-directed enzyme prodrug therapy); the formerinvolving targeting the activating agent to the cells by conjugation toa cell-specific antibody, while the latter involves producing theactivating agent, e.g. a JNK inhibitor sequence or the chimeric peptide,in a vector by expression from encoding DNA in a viral vector (see forexample, EP-A-415731 and WO 90/07936).

According to a further embodiment, the JNK inhibitor sequences, chimericpeptides, nucleic acid sequences or antibodies to JNK inhibitorsequences or to chimeric peptides as defined herein, e.g. an JNKinhibitor sequence according to any of sequences of SEQ ID NOs: 1 to 4and 13 to 20 and 33-100 and/or a chimeric peptide according to any ofsequences of SEQ ID NOs: 9 to 12 and 23 to 32, and/or an JNK inhibitorsequence according to any of sequences of SEQ ID NOs: 1 to 4 and 13 to20 and 33-100 comprising a trafficking sequence according to any of SEQID NOs: 5 to 8 and 21 to 22, or variants or fragments thereof within theabove definitions, may be utilized in (in vitro) assays (e.g.immunoassays) to detect, prognose, diagnose, or monitor variousconditions and disease states selected from non-chronic or chronicinflammatory digestive diseases as defined above, or monitor thetreatment thereof. The immunoassay may be performed by a methodcomprising contacting a sample derived from a patient with an antibodyto an JNK inhibitor sequence, a chimeric peptide, or a nucleic acidsequence, as defined above, under conditions such thatimmunospecific-binding may occur, and subsequently detecting ormeasuring the amount of any immunospecific-binding by the antibody. In aspecific embodiment, an antibody specific for an JNK inhibitor sequence,a chimeric peptide or a nucleic acid sequence may be used to analyze atissue or serum sample from a patient for the presence of JNK or a JNKinhibitor sequence; wherein an aberrant level of JNK is indicative of adiseased condition. The immunoassays that may be utilized include, butare not limited to, competitive and non-competitive assay systems usingtechniques such as Western Blots, radioimmunoassays (RIA), enzyme linkedimmunosorbent assay (ELISA), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,fluorescent immunoassays, complement-fixation assays, immunoradiometricassays, and protein-A immunoassays, etc. Alternatively, (in vitro)assays may be performed by delivering the JNK inhibitor sequences,chimeric peptides, nucleic acid sequences or antibodies to JNK inhibitorsequences or to chimeric peptides, as defined above, to target cellstypically selected from e.g. cultured animal cells, human cells ormicro-organisms, and to monitor the cell response by biophysical methodstypically known to a skilled person. The target cells typically usedtherein may be cultured cells (in vitro) or in vivo cells, i.e. cellscomposing the organs or tissues of living animals or humans, ormicroorganisms found in living animals or humans.

The present invention additionally provides the use of kits fordiagnostic or therapeutic purposes, particular for the treatment,prevention or monitoring of non-chronic or chronic inflammatorydigestive diseases as defined above, wherein the kit includes one ormore containers containing JNK inhibitor sequences, chimeric peptides,nucleic acid sequences and/or antibodies to these JNK inhibitorsequences or to chimeric peptides as defined above, e.g. an anti-JNKinhibitor sequence antibody to an JNK inhibitor sequence according toany of sequences of SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100, to achimeric peptide according to any of sequences of SEQ ID NOs: 9 to 12and 23 to 32, to an JNK inhibitor sequence according to any of sequencesof SEQ ID NOs: 1 to 4 and 13 to 20 and 33-100 comprising a traffickingsequence according to any of SEQ ID NOs: 5 to 8 and 21 to 22, or to orvariants or fragments thereof within the above definitions, or such ananti-JNK inhibitor sequence antibody and, optionally, a labeled bindingpartner to the antibody. The label incorporated thereby into theantibody may include, but is not limited to, a chemiluminescent,enzymatic, fluorescent, colorimetric or radioactive moiety. In anotherspecific embodiment, kits for diagnostic use in the treatment,prevention or monitoring of non-chronic or chronic inflammatorydigestive diseases as defined above are provided which comprise one ormore containers containing nucleic acids that encode, or alternatively,that are the complement to, an JNK inhibitor sequence and/or a chimericpeptide as defined above, optionally, a labeled binding partner to thesenucleic acids, are also provided. In an alternative specific embodiment,the kit may be used for the above purposes as a kit, comprising one ormore containers, a pair of oligonucleotide primers (e.g. each 6-30nucleotides in length) that are capable of acting as amplificationprimers for polymerase chain reaction (PCR; see e.g. Innis, et al, 1990.PCR PROTOCOLS, Academic Press, Inc., San Diego, Calif.), ligase chainreaction, cyclic probe reaction, and the like, or other methods knownwithin the art used in context with the nucleic acids as defined above.The kit may, optionally, further comprise a predetermined amount of apurified JNK inhibitor sequence as defined above, a chimeric peptide asdefined above, or nucleic acids encoding these, for use as a diagnostic,standard, or control in the assays for the above purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

DESCRIPTION OF FIGURES

FIGS. 1A-C are diagrams showing alignments of conserved JBD domainregions in the indicated transcription factors. JNK inhibitor sequencesused herein were identified by carrying out sequence alignments. Theresults of this alignment are exemplarily shown in FIGS. 1A-1C. FIG. 1Adepicts the region of highest homology between the JBDs of IB1, IB2,c-Jun and ATF2. Panel B depicts the amino acid sequence of the JBDs ofL-IB1(s) and L-IB1 for comparative reasons. Fully conserved residues areindicated by asterisks, while residues changed to Ala in theGFP-JBD_(23Mut) vector are indicated by open circles. FIG. 1C shows theamino acid sequences of chimeric proteins that include a JNK inhibitorsequence and a trafficking sequence. In the example shown, thetrafficking sequence is derived from the human immunodeficiency virus(HIV) TAT polypeptide, and the JNK inhibitor sequence is derived from anIB1(s) polypeptide. Human, mouse, and rat sequences are identical inPanels B and C.

FIG. 2 is a diagram showing sequences of generic TAT-IB fusion peptidesfrom human, mouse and rat.

FIG. 3 shows the clinical scores upon treatment with XG-102 (SEQ ID NO:11) in an IBD study with a treatment using XG-102 in a concentration of1 and 100 μg/kg SC daily.

FIG. 4 shows a dose response curve upon treatment with XG-102 (SEQ IDNO: 11) in an IBD study with a treatment using XG-102, in aconcentration of 0.01, 0.1, 1, 10, 100 and 1000 μg/kg SC daily.

FIG. 5 shows the clinical scores upon treatment with XG-102 (SEQ ID NO:11) in an IBD study with a treatment using XG-102 (single dose SC) in aconcentration of 1 and 100 μg/kg SC as a single dose on day 0.

FIG. 6 shows the clinical scores upon treatment with XG-102 (SEQ ID NO:11) in an IBD study with a treatment using XG-102 (daily, PO) in aconcentration of 1 and 100 μg/kg PO as a repeated dose.

FIG. 7 shows the clinical scores upon treatment with XG-102 (SEQ ID NO:11) in an IBD study with a treatment using XG-102 (single dose PO) in aconcentration of 1 and 100 μg/kg PO as a single dose on day 0.

FIG. 8 Primary cultured macrophages were incubated with XG-102 (SEQ IDNO: 11) and extensively washed. Presence of XG-102 (SEQ ID NO: 11) wasrevealed using a specific antibody against XG-102. XG-102 is stronglyincorporated into primary macrophages.

FIG. 9 Mice were treated via three different routes of administration(s.c., i.v., i.p.) with radiolabeled peptides with C¹⁴ (1 mg/kg).Animals were sacrificed 72 hours after injection and processed forimmunoradiography. Sagital sections were exposed and revealed theaccumulation XG-102 peptides in the liver, spleen, and bone marrowpredominantly (XG-102: SEQ ID NO: 11).

FIG. 10 shows an immunostaining against XG-102 (SEQ ID NO: 11) in theliver of rats injected with 1 mg/kg of XG-102 i.v. Animals weresacrificed 24 hours after injection. Revelation was done using DABsubstrate. This figure shows again the pronounced accumulation of XG-102in the liver, and especially, in the Kupffer cells (macrophages).

FIG. 11 shows the inhibition of Cytokine & Chemokine Release in two celllines. XG-102 (SEQ ID NO:11) inhibits cytokine release in both myeloidand lymphoid cell lines, reducing LPS-induced TNFa, IL-6 and MCP-1release in THP-1 cells (Panels A-C) and PMA & ionomycin-induced IFNg,IL-6 and IL-2 production in Jurkat cells (Panels D-F). The control(XG-101) is less effective due to its lesser stability.

FIG. 12 shows the inhibition of cytokine release in primary cells.XG-102 (SEQ ID NO:11) also inhibits cytokine release in primary lymphoidand myeloid cells, reducing LPS-induced TNFa, IL-6 and Rantes release inmurine macrophages (Panels A-C) and PMA & ionomycin-induced TNFa andIFNg production in murine T cells (Panels D-E). Effects occur atnon-cytotoxic concentrations of XG-102 (Panel F)

FIG. 13 shows the effect in TNBS-induced Colitis. JNK is activated inmacrophages and lymphocytes of patients with inflammatory bowel disease,a response correlated with increased TNFa, IL-6 and IFNg production inlesions. Subcutaneous administration of 50 and 100 mg/kg XG-102 protectsmice from TNBS-induced colitis (Panels A-C), diminishing DAI, weightloss and rectal bleeding.

FIG. 14 shows the IB1 cDNA sequence from rat and its predicted aminoacid sequence (SEQ ID NO:102)

FIG. 15 shows the IB1 protein sequence from rat encoded by theexon-intron boundary of the rIB1 gene-splice donor (SEQ ID NO:103)

FIG. 16 shows the IB1 protein sequence from Homo sapiens (SEQ ID NO:104)

FIG. 17 shows the IB1 cDNA sequence from Homo sapiens (SEQ ID NO:105)

EXAMPLES Example 1 Identification of JNK Inhibitor Sequences

Amino acid sequences important for efficient interaction with JNK wereidentified by sequence alignments between known JNK binding domain JBDs.A sequence comparison between the JBDs of IB1 [SEQ ID NO: 13], IB2 [SEQID NO: 14], c-Jun [SEQ ID NO: 15] and ATF2 [SEQ ID NO: 16] defined aweakly conserved 8 amino acid sequence (FIG. 1A). Since the JBDs of IB1and IB2 are approximately 100 fold as efficient as c-Jun or ATF2 inbinding JNK (Dickens et al. Science 277: 693 (1997), it was reasonedthat conserved residues between IB1 and IB2 must be important to confermaximal binding. The comparison between the JBDs of IB1 and IB2 definedtwo blocks of seven and three amino acids that are highly conservedbetween the two sequences.

These two blocks are contained within a peptide sequence of 19 aminoacids in L-IB1(s) [SEQ ID NO: 1] and are also shown for comparativereasons in a 23 aa peptide sequence derived from IB1 [SEQ ID NO: 17].These sequences are shown in FIG. 1B, dashes in the L-IB1 sequenceindicate a gap in the sequence in order to align the conserved residueswith L-IB1(s).

Example 2 Preparation of JNK Inhibitor Fusion Proteins

JNK inhibitor fusion proteins according to SEQ ID NO: 9 were synthesizedby covalently linking the C-terminal end of SEQ ID NO: 1 to a N-terminal10 amino acid long carrier peptide derived from the HIV-TAT4g 57 (Viveset al., J. Biol. Chem. 272: 16010 (1997)) according to SEQ ID NO: 5 viaa linker consisting of two proline residues. This linker was used toallow for maximal flexibility and prevent unwanted secondary structuralchanges. The basic constructs were also prepared and designated L-IB1(s)(SEQ ID NO: 1) and L-TAT [SEQ ID NO: 5], respectively.

All-D retro-inverso peptides according to SEQ ID NO: 11 were synthesizedaccordingly. The basic constructs were also prepared and designatedD-IB1(s) [SEQ ID NO: 2] and D-TAT [SEQ ID NO: 6], respectively.

All D and L fusion peptides according to SEQ ID NOs: 9, 10, 11 and 12were produced by classical Fmock synthesis and further analysed by MassSpectrometry. They were finally purified by HPLC. To determine theeffects of the proline linker, two types of TAT peptide were producedone with and one without two prolines. The addition of the two prolinesdid not appear to modify the entry or the localization of the TATpeptide inside cells. Generic peptides showing the conserved amino acidresidues are given in FIG. 2.

Example 3 Inhibition of Cell Death by JBD19

Effects of the 19 aa long JBD sequence of IB1(s) on JNK biologicalactivities were studied. The 19 aa sequence was linked N-terminal to theGreen Fluorescent Protein (GFP JBD19 construct), and the effect of thisconstruct on pancreatic beta-cell apoptosis induced by IL1 wasevaluated. This mode of apoptosis was previously shown to be blocked bytransfection with JBD₁₋₂₈₀ whereas specific inhibitors of ERK½ or p38did not protect (see Ammendrup et al., supra).

Oligonucleotides corresponding to JBD19 and comprising a conservedsequence of 19 amino acids as well as a sequence mutated at the fullyconserved regions were synthesized and directionally inserted into theEcoRI and SalI sites of the pEGFP-N1 vector encoding the GreenFluorescent Protein (GFP) (from Clontech). Insulin producing TC-3 cellswere cultured in RPMI 1640 medium supplemented with 10% Fetal CalfSerum, 100 μg/mL Streptomycin, 100 units/mL Penicillin and 2 mMGlutamine. Insulin producing TC-3 cells were transfected with theindicated vectors and IL-1 (10 ng/mL) was added to the cell culturemedium. The number of apoptotic cells was counted at 48 hours after theaddition of IL-1 using an inverted fluorescence microscope. Apoptoticcells were discriminated from normal cells by the characteristic“blebbing out” of the cytoplasm and were counted after two days.

GFP is Green Fluorescent protein expression vector used as a control;JBD19 is the vector expressing a chimeric GFP linked to the 19 aasequence derived from the JBD of IB1; JBD19Mut is the same vector asGFP-JBD19, but with a JBD mutated at four conserved residues shown asFIG. 1B; and JBD₁₋₂₈₀ is the GFP vector linked to the entire JBD (aa1-280). The GFP-JBD19 expressing construct prevented IL-1 inducedpancreatic-cell apoptosis as efficiently as the entire JBD₁₋₂₈₀.

As additional controls, sequences mutated at fully conserved IB1(s)residues had greatly decreased ability to prevent apoptosis.

Example 4 Cellular Import of TAT-IB1(s) Peptides

The ability of the L- and D-enantiomeric forms of TAT and TAT-IB1(s)peptides (“TAT-IB peptides”) to enter cells was evaluated. L-TAT, D-TAT,L-TAT-IB1(s), and D-TAT-IB1(s) peptides [SEQ ID NOs: 5, 6, 9 and 12,respectively] were labeled by N-terminal addition of a glycine residueconjugated to fluorescein. Labeled peptides (1 μM) were added to TC-3cell cultures, which were maintained as described in Example 3. Atpredetermined times cells were washed with PBS and fixed for fiveminutes in ice-cold methanol-acetone (1:1) before being examined under afluorescence microscope. Fluorescein-labeled BSA (1 μM, 12 moles/moleBSA) was used as a control. Results demonstrated that all the abovefluorescein labeled peptides had efficiently and rapidly (less than fiveminutes) entered cells once added to the culture medium. Conversely,fluorescein labeled bovine serum albumin (1 μM BSA, 12 molesfluorescein/mole BSA) did not enter the cells.

A time course study indicated that the intensity of the fluorescentsignal for the L-enantiomeric peptides decreased by 70% following a 24hours period. Little to no signal was present at 48 hours. In contrast,D-TAT and D-TAT-IB1(s) were extremely stable inside the cells.

Fluorescent signals from these all-D retro-inverso peptides were stillvery strong 1 week later, and the signal was only slightly diminished at2 weeks post treatment.

Example 5 In Vitro Inhibition of c-JUN, ATF2 and Elk1 Phosphorylation

The effects of the peptides on JNKs-mediated phosphorylation of theirtarget transcription factors were investigated in vitro. Recombinant andnon activated JNK1, JNK2 and JNK3 were produced using a TRANSCRIPTIONAND TRANSLATION rabbit reticulocyte lysate kit (Promega) and used insolid phase kinase assays with c-Jun, ATF2 and Elk1, either alone orfused to glutathione-S-transferase (GST), as substrates. Dose responsestudies were performed wherein L-TAT or L-TAT-IB1(s) peptides (0-25 μM)were mixed with the recombinant JNK1, JNK2, or JNK3 kinases in reactionbuffer (20 mM Tris-acetate, 1 mM EGTA, 10 mM p-nitrophenyl-phosphate(pNPP), 5 mM sodium pyrophosphate, 10 mM p-glycerophosphate, 1 mMdithiothreitol) for 20 minutes. The kinase reactions were then initiatedby the addition of 10 mM MgCl₂ and 5 pCi ³³P-dATP and 1 μg of eitherGST-Jun (aa 1-89), GST-AFT2 (aa 1-96) or GST-ELK1 (aa 307-428).GST-fusion proteins were purchased from Stratagene (La Jolla, Calif.).

Ten μL of glutathione-agarose beads were also added to the mixture.Reaction products were then separated by SDS-PAGE on a denaturing 10%polyacrylamide gel. Gels were dried and subsequently exposed to X-rayfilms (Kodak). Nearly complete inhibition of c-Jun, ATF2 and Elk1phosphorylation by JNKs was observed at TAT-IB(s) peptide doses as lowas 2.5 μM. However, a marked exception was the absence of TAT-IB(s)inhibition of JNK3 phosphorylation of Elk1. Overall, the TAT-IB1(s)peptide showed superior effects in inhibiting JNK family phosphorylationof their target transcription factors. The ability of D-TAT,D-TAT-IB1(s) and L-TAT-IB1(s) peptides (0-250 μM dosage study) toinhibit GST-Jun (aa 1-73) phosphorylation by recombinant JNK1, JNK2, andJNK3 by were analyzed as described above. Overall, D-TAT-IB1(s) peptidedecreased JNK-mediated phosphorylation of c-Jun, but at levelsapproximately 10-20 fold less efficiently than L-TAT-IB1(s).

Example 6 Inhibition of c-JUN Phosphorylation by Activated JNKs

The effects of the L-TAT or L-TAT-IB1(s) peptides as defined herein onJNKs activated by stressful stimuli were evaluated using GST-Jun to pulldown JNKs from UV-light irradiated HeLa cells or IL-1 treated PTC cells.PTC cells were cultured as described above. HeLa cells were cultured inDMEM medium supplemented with 10% Fetal Calf Serum, 100 μg/mLStreptomycin, 100 units/ml Penicillin and 2 mM Glutamine. One hour priorto being used for cell extract preparation, PTC cells were activatedwith IL-1 as described above, whereas HeLa cells were activated byUV-light (20 J/m²). Cell extracts were prepared from control, UV-lightirradiated HeLa cells and IL-1 treated TC-3 cells by scraping the cellcultures in lysis buffer (20 mM Tris-acetate, 1 mM EGTA, 1% TritonX-100, 10 mM p-nitrophenyl-phosphate, 5 mM sodium pyrophosphate, 10mMP-glycerophosphate, 1 mM dithiothreitol). Debris were removed bycentrifugation for five minutes at 15,000 rpm in an SS-34 Beckman rotor.One-hundred μg extracts were incubated for one hour at room temperaturewith one μg GST-jun (amino acids 1-89) and 10 μL of glutathione-agarosebeads (Sigma). Following four washes with the scraping buffer, the beadswere resuspended in the same buffer supplemented with L-TAT orL-TAT-IB1(s) peptides (25 μM) for 20 minutes. Kinase reactions were theninitiated by addition of 10 mM MgCl₂ and 5 pCi ³³P-gamma-dATP andincubated for 30 minutes at 30° C.

Reaction products were then separated by SDS-PAGE on a denaturing 10%polyacrylamide gel. Gels were dried and subsequently exposed to X-rayfilms (Kodak). The TAT-IB(s) peptides efficiently preventedphosphorylation of c-Jun by activated JNKs in these experiments.

Example 7 In Vivo Inhibition of c-JUN Phosphorylation by TAT-Ib(s)Peptides as Defined Herein

To determine whether the cell-permeable peptides as defined herein couldblock JNK signaling in vivo, we used a heterologous GAL4 system. HeLacells, cultured as described above, were co-transfected with the5×GAL-LUC reporter vector together with the GAL-Jun expression construct(Stratagene) comprising the activation domain of c-Jun (amino acids1-89) linked to the GAL4 DNA-binding domain. Activation of JNK wasachieved by the co-transfection of vectors expressing the directlyupstream kinases MKK4 and MKK7 (see Whitmarsh et al, Science 285: 1573(1999)). Briefly, 3×10⁵ cells were transfected with the plasmids in3.5-cm dishes using DOTAP (Boehringer Mannheim) following instructionsfrom the manufacturer. For experiments involving GAL-Jun, 20 ng of theplasmid was transfected with 1 μg of the reporter plasmid pFR-Luc(Stratagene) and 0.5 μg of either MKK4 or MKK7 expressing plasmids.Three hours following transfection, cell media were changed and TAT andTAT-IB1(s) peptides (1 μM) were added. The luciferase activities weremeasured 16 hours later using the “Dual Reporter System” from Promegaafter normalization to protein content. Addition of TAT-IB1(s) peptideblocked activation of c-Jun following MKK4 and MKK7 mediated activationof JNK. Because HeLa cells express JNK1 and JNK2 isoforms but not JNK3,we transfected cells with JNK3. Again, the TAT-IB(s) peptide inhibitedJNK2 mediated activation of c-Jun.

Example 8 Synthesis of all-D Retro-Inverso IB(s) Peptides and VariantsThereof

Peptides of the invention may be all-D amino acid peptides synthesizedin reverse to prevent natural proteolysis (i.e. all-D retro-inversopeptides). An all-D retro-inverso peptide of the invention would providea peptide with functional properties similar to the native peptide,wherein the side groups of the component amino acids would correspond tothe native peptide alignment, but would retain a protease resistantbackbone.

Retro-inverso peptides of the invention are analogs synthesized usingD-amino acids by attaching the amino acids in a peptide chain such thatthe sequence of amino acids in the retro-inverso peptide analog isexactly opposite of that in the selected peptide which serves as themodel. To illustrate, if the naturally occurring TAT protein (formed ofL-amino acids) has the sequence GRKKRRQRRR [SEQ ID NO: 5], theretro-inverso peptide analog of this peptide (formed of D-amino acids)would have the sequence RRRQRRKKRG [SEQ ID NO: 6]. The procedures forsynthesizing a chain of D-amino acids to form the retro-inverso peptidesare known in the art (see e.g. Jameson et al, Nature, 368, 744-746(1994); Brady et al., Nature, 368, 692-693 (1994); Guichard et al., J.Med. Chem. 39, 2030-2039 (1996)). Specifically, the retro-peptidesaccording to SEQ ID NOs 2, 4, 6, 8, 11-12, 18, 20, 22 and 25-26, wereproduced by classical F-mock synthesis and further analyzed by MassSpectrometry. They were finally purified by HPLC.

Since an inherent problem with native peptides is degradation by naturalproteases and inherent immunogenicity, the heterobivalent orheteromultivalent compounds of this invention will be prepared toinclude the “retro-inverso isomer” of the desired peptide. Protectingthe peptide from natural proteolysis should therefore increase theeffectiveness of the specific heterobivalent or heteromultivalentcompound, both by prolonging half-life and decreasing the extent of theimmune response aimed at actively destroying the peptides.

Example 9 Long Term Biological Activity of all-D Retro-Inverso IB(s)Peptides and Variants Thereof

Long term biological activity is predicted for the D-TAT-IB(s)retro-inverso containing peptide heteroconjugate (see chimeric sequencesabove) when compared to the native L-amino acid analog owing toprotection of the D-TAT-IB(s) peptide from degradation by nativeproteases, as shown in Example 5.

Inhibition of IL-1 induced pancreatic beta-cell death by theD-TAT-IB1(s) peptide was analyzed. TC-3 cells were incubated asdescribed above for 30 minutes with one single addition of the indicatedpeptides (1, μM), then IL-1 (10 ng/ml) was added.

Apoptotic cells were then counted after two days of incubation with IL-1by use of Propidium Iodide and Hoechst 33342 nuclear staining. A minimumof 1,000 cells were counted for each experiment. The D-TAT-IB1 peptidedecreased IL-1 induced apoptosis to a similar extent as L-TAT-IBpeptides.

Long term inhibition of IL-1P induced cell-death by the D-TAT-IB1peptide was also analyzed. TC-3 cells were incubated as above for 30minutes with one single addition of the indicated peptides (1 μM), thenIL-1 (10 ng/ml) was added, followed by addition of the cytokine everytwo days. Apoptotic cells were then counted after 15 days of incubationwith IL-1 by use of propidium iodide and Hoechst 33342 nuclear staining.Note that one single addition of the TAT-IB1 peptide does not conferlong-term protection. A minimum of 1.000 cells were counted for eachexperiment. As a result, D-TAT-IB1(s), but not L-TAT-IB1(s), was able toconfer long term (15 day) protection.

Example 10 Suppression of JNK Transcription Factors by L-TAT-IB1(s)Peptides as Used According to the Present Invention

Gel retardation assays were carried out with an AP-1 doubled labeledprobe (5′-CGC TTG ATG AGT CAG CCG GAA-3′ (SEQ ID NO: 101). HeLa cellnuclear extracts that were treated or not for one hour with 5 ng/mlTNF-,as indicated. TAT and L-TAT-IB1(s) peptides as used according to thepresent invention were added 30 minutes before TNF-alpha. Only the partof the gel with the specific AP-1 DNA complex (as demonstrated bycompetition experiments with non-labeled specific and non-specificcompetitors) is shown.

L-TAT-IB1(s) peptides as used according to the present inventiondecrease the formation of the AP-1 DNA binding complex in the presenceof TNF-alpha.

Example 11 Inhibition of Endogenous JNK Activity in HepG2 Cells Using anall-in One Well Approach

HepG2 cells were seeded at 3′000 cells/well the day prior theexperiment. Then, increasing concentrations of either interleukin-1[IL-1beta)] or tumor necrosis factor [TNFalpha)] (a) were added toactivate JNK for 30 minutes. Cells were lysed in 20 mM Hepes, 0.5% TweenpH 7.4 and processed for AlphaScreen JNK. (b) Z′ for the JNK activityinduced by 10 ng/ml IL-1 and measured in 384 wells/plate (n=96). (c)Inhibition of endogenous IL-1 beta-induced JNK activity with chemicalJNK inhibitors [staurosporin and SP600125]. (d) Effect of peptidicinhibitors L-TAT-IB1(s) according to SEQ ID NO: 9 [here abbreviated asL-JNKi) and D-TAT-IB1(s) according to SEQ ID NO: 11 (here abbreviated asD-JNKi) and JBDs) (corresponds to L-JNKI without the TAT sequence)] onIL-1 dependent JNK activity. All panels are representative of threeindependent experiments (n=3).

Methods: Alphascreen Kinase Assay

Principle: AlphaScreen is a non-radioactive bead-based technology usedto study biomolecular interactions in a microplate format. The acronymALPHA stands for Amplified Luminescence Proximity Homogenous Assay. Itinvolves a biological interaction that brings a “donor” and an“acceptor” beads in close proximity, then a cascade of chemicalreactions acts to produce an amplified signal. Upon laser excitation at680 nm, a photosensitizer (phthalocyanine) in the “donor” bead convertsambient oxygen to an excited singlet state. Within its 4 μsec half-life,the singlet oxygen molecule can diffuse up to approximately 200 nm insolution and if an acceptor bead is within that proximity, the singletoxygen reacts with a thioxene derivative in the “acceptor” bead,generating chemiluminescence at 370 nm that further activatesfluorophores contained in the same “acceptor” bead. The excitedfluorophores subsequently emit light at 520-620 nm. In the absence of anacceptor bead, singlet oxygen falls to ground state and no signal isproduced.

Kinase reagents (B-GST-cJun, anti P-cJun antibody and active JNK3) werefirst diluted in kinase buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 1 mMDTT, 100 μM Na₃VO₄, 0.01% Tween-20) and added to wells (15 μl).Reactions were then incubated in presence of 10 μM of ATP for 1 h at 23°C. Detection was performed by an addition of 10 μl of beads mix (ProteinA acceptor 20 μg/ml and Streptavidin donor 20 μg/ml), diluted indetection buffer (20 mM Tris-HCl pH 7.4, 20 mM NaCl, 80 mM EDTA, 0.3%BSA), followed by an another one-hour incubation at 23° C. in the dark.For measurement of JNK endogenous activity, kinase assays were performedas described above except active JNK3 was replaced by cells lysates andreaction kinase components were added after the cells lysis. B-GST-cjunand P-cJun antibody were used at the same concentrations whereas ATP wasused at 50 μM instead of 10 μM. AlphaScreen signal was analyzed directlyon the Fusion or En Vision apparatus.

Example 12 Evaluation of the Therapeutical Activity of D- andL-TAT-IB1(s) Peptides as Used According to the Present Invention

a) Test system:

-   -   i) Species/Strain: Mouse/BALB/c    -   ii) Source: Harlan Israel, Ltd.    -   iii) Gender: Female    -   iv) Total No. of Animals: n=150    -   v) Age: Young adults, 7 weeks of age at study initiation    -   vi) Body Weight: Weight variation of animals at the time of        treatment initiation does not exceed ±20% of the mean weight.    -   vii) Animals Health: The health status of the animals used in        this study is examined on arrival, only animals in good health        are acclimatized to laboratory conditions (at least seven days)        and are used in the study.    -   viii) Randomization: Animals are randomly assigned to        experimental groups according to a Table of Random Numbers.    -   ix) Termination: At the end of the study surviving animals are        euthanized by cercical dislocation.

b) Constitution of test groups and dose levels

-   -   The table below lists the experimental groups comprising the        study.

Group Group Test Volume # size Item Route Dose (ml/kg) Regime 1F N = 10Vehicle PO 0 5 Once daily for 7 days 2F N = 10 Sulfasalazine PO 10 mg/kg5 Once daily for 7 days 3F N = 10 Remicade IP 5 mg/kg 5 Once daily for 7days 4F N = 10 XG-102 SC 0.01 μg/kg 5 Once daily for 7 days 5F N = 10XG-102 SC 0.1 μg/kg 5 Once daily for 7 days 6F N = 10 XG-102 SC 1 μg/kg5 Once daily for 7 days 7F N = 10 XG-102 SC 10 μg/kg 5 Once daily for 7days 8F N = 10 XG-102 SC 100 μg/kg 5 Once daily for 7 days 9F N = 10XG-102 SC 1000 μg/kg 5 Once daily for 7 days 10F  N = 10 XG-102 SC 1μg/kg 5 Single dose 11F  N = 10 XG-102 SC 100 μg/kg 5 Single dose 12F  N= 10 XG-102 PO 1 μg/kg 5 Once daily for 7 days 13F  N = 10 XG-102 PO 100μg/kg 5 Once daily for 7 days 14F  N = 10 XG-102 PO 1 μg/kg 5 Singledose 15F  N = 10 XG-102 PO 100 μg/kg 5 Single dose XG-102 = SEQ ID NO:11 IP = intraperitoneal administration PO = peroral administration SC =subcutaneous administration

c) Test Procedures

-   -   Colitis was induced by administration of TNBS dissolved in 50%        Ethanol    -   All animals were then treated with doses of XG-102 in the range        of 0.1 to 1000 μg/kg, either intraperitoneally or        subcutaneously, as a single or repeated daily doses (see above).

d) Observations and Examinations

-   -   i) Clinical signs        -   Throughout the duration of the above experiment, careful            clinical examinations were carried out and recorded.            Observations included changes external appearance, e.g. of            the skin, fur, eyes, mucous membranes, occurrence of            secretions and excretions (e.g. diarrhea), and autonomic            activity. Changes in gait, posture and response to handling,            as well as the presence of bizarre behavior, tremors,            convulsions, sleep and coma were also noted.    -   ii) Body weights        -   Determination of individual body weight of animals was made            on a daily basis.    -   iii) Clinical assessment of colitis        -   Body weight, stool consistency and bleeding per rectum were            all recorded daily and served as the parameters of disease            severity score:

Presence of Score Weight loss (%) Stool consistency blood per rectum 0None Normal Negative 1 1-5 Redness, swelling Negative of the anus 2 5-10 Loose stool Negative 3 10-15 Diarrhea Negative 4 >15 DiarrheaBleeding 5 Death

-   -   iv) Gross pathology of the colon        -   On the last day of the experiment, animals were euthanized            and the colon was removed for gross pathology evaluation            according to the following score:

Grade Signs 0 No abnormalities detected 1 Edema and redness on onelocation 2 Edema and redness on more than one location, or a verymassive endema and redness capture more than 50% of the colon 3 Oneulcer 4 More than one ulcer or a very long severe ulcer

e) Results

-   -   i) Clinical signs        -   No abnormalities were observed during clinical examinations            following the treatment with XG-102 (SEQ ID NO: 11).    -   ii) Mortality rate        -   No mortality was recorded.    -   iii) Body weights        -   TNBS induced a significant weight loss on day 1. XG-102 (SEQ            ID NO: 11) administration either prevented the weight loss            or ameliorated the symptoms and supported recovery.    -   iv) Clinical score        -   TNBS injected vehicle treated animals reached a maximum            score on study day 1 and recovered fully only on or after            study day 5. FIG. 3 shows that Sulfasalazine treatment            resulted in reduction in the clinical score. FIGS. 3-7            demonstrate that XG-102 (SEQ ID NO: 11), administered using            any dose, route or time schedule as defined above (single            dose or daily dose) resulted in an effect equivalent to or            better than the one observed with the commonly used            reference drug sulfasalazine.    -   v) Cross pathology score        -   Gross analysis at the end of the study revealed that the            TNBS injected vehicle treated animals were injured with            edema and ulcers along the colon. Sulfasalazine was            effective in reducing the gross pathology completely.    -   vi) Colon length        -   No effect of disease induction or treatment was observed on            colon length.    -   vii) Colon weight        -   No effect of disease induction or treatment was observed on            colon weight.

f) Conclusions

-   -   In view of the above findings obtained under the conditions of        the above experiment and confined to the in-life data, the        exemplary sequence XG-102 according to SEQ ID NO: 11        administered either SC or PO was active in enhancing diseases        recovery.

Example 13 Preferred Embodiments

In the following, some preferred embodiments according to the presentinvention are listed:

-   1. Use of a JNK inhibitor sequence comprising less than 150 amino    acids in length for the preparation of a pharmaceutical composition    for treating chronic or non-chronic inflammatory digestive diseases    in a subject.-   2. The use of a JNK inhibitor sequence according to embodiment 1,    wherein the JNK inhibitor sequence is derived from a human or rat    IB1 sequence as defined or encoded by any of sequences according to    SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 or    from a fragment or variant thereof.-   3. The use of a JNK inhibitor sequence according to embodiment 1 or    2, wherein the JNK inhibitor sequence comprises a range of 5 to 150    amino acid residues, more preferably 10 to 100 amino acid residues,    even more preferably 10 to 75 amino acid residues and most    preferably a range of 10 to 50 amino acid residues.-   4. The use of a JNK inhibitor sequence of any of embodiments 1 to 3,    wherein the JNK inhibitor sequence binds c-jun amino terminal kinase    (JNK).-   5. The use of a JNK inhibitor sequence of any of embodiments 1 to 4,    wherein the JNK inhibitor sequence inhibits the activation of at    least one JNK targeted transcription factor when the JNK inhibitor    sequence is present in a JNK expressing cell.-   6. The use of a JNK inhibitor sequence of any of embodiments 1 to 5,    wherein the JNK targeted transcription factor is selected from the    group consisting of c-Jun, ATF2, and Elk1.-   7. The use of a JNK inhibitor sequence of any of embodiments 1 to 6,    wherein the JNK inhibitor sequence alters a JNK effect when the    peptide is present in a JNK expressing cell.-   8. The use of a JNK inhibitor sequence of any of embodiments 1 to 7,    wherein the JNK inhibitor sequence is composed of L-amino acids,    D-amino acids, or a combination of both, preferably comprises at    least 1 or even 2, preferably at least 3, 4 or 5, more preferably at    least 6, 7, 8 or 9 and even more preferably at least 10 or more D-    and/or L-amino acids, wherein the D- and/or L-amino acids may be    arranged in the JNK inhibitor sequences in a blockwise, a    non-blockwise or in an alternate manner.-   9. The use of a JNK inhibitor sequence of any of embodiments 1 to 8,    wherein the inhibitor sequence comprises or consists of at least one    amino acid sequence according to SEQ ID NOs: 1 to 4, 13 to 20 and 33    to 100, or a fragment, derivative or variant thereof.-   10. Use of a chimeric peptide comprising at least one first domain    and at least one second domain linked by a covalent bond, the first    domain comprising a trafficking sequence, and the second domain    comprising a JNK inhibitor sequence as defined in any of embodiments    1 to 9 for the preparation of a pharmaceutical composition for    treating chronic or non-chronic inflammatory digestive diseases in a    subject.-   11. The use of the chimeric peptide of embodiment 10, wherein the    chimeric peptide is composed of L-amino acids, D-amino acids, or a    combination of both, preferably comprises at least 1 or even 2,    preferably at least 3, 4 or 5, more preferably at least 6, 7, 8 or 9    and even more preferably at least 10 or more D- and/or L-amino    acids, wherein the D- and/or L-amino acids may be arranged in the    chimeric peptide in a blockwise, a non-blockwise or in an alternate    manner.-   12. The use of the chimeric peptide of embodiment 10 or 11, wherein    the trafficking sequence comprises the amino acid sequence of a    human immunodeficiency virus TAT polypeptide.-   13. The use of the chimeric peptide of any of embodiments 10 to 12,    wherein the trafficking sequence consists of or comprises the amino    acid sequence of SEQ ID NO: 5, 6, 7, 8, 21 or 22.-   14. The use of the chimeric peptide of any of embodiments 10 to 13,    wherein the trafficking sequences augments cellular uptake of the    peptide.-   15. The use of the chimeric peptide of any of embodiments 10 to 14,    wherein the trafficking sequence directs nuclear localization of the    peptide.-   16. The use of the chimeric peptide of any of embodiments 10 to 15,    wherein the chimeric peptide consists of or comprises the amino acid    sequence of any of SEQ ID NOs: 9 to 12 and 23 to 32, or a fragment,    or variant thereof.-   17. Use of an isolated nucleic acid encoding a JNK inhibitor    sequence as defined in any of embodiments 1 to 9 or a chimeric    peptide as defined in any of embodiments 10 to 16 for the    preparation of a pharmaceutical composition for treating chronic or    non-chronic inflammatory digestive diseases in a subject.-   18. Use of a vector comprising the nucleic acid as defined in    embodiment 17 for the preparation of a pharmaceutical composition    for treating chronic or non-chronic inflammatory digestive diseases    in a subject.-   19. Use of a cell comprising the vector as defined in embodiment 18    for the preparation of a pharmaceutical composition for treating    chronic or non-chronic inflammatory digestive diseases in a subject.-   20. Use of an antibody which binds immunospecifically to a JNK    inhibitor sequence according to any of embodiments 1 to 9 or to a    chimeric peptide according to any of embodiments 10 to 16 for the    preparation of a pharmaceutical composition for treating chronic or    non-chronic inflammatory digestive diseases in a subject.-   21. Use according to any of the preceding embodiments, wherein the    pharmaceutical composition is to be administered by an    administration route selected from the group consisting of    parenteral routes, including intravenous, intramuscular,    subcutaneous, intradermal, transdermal, enteral routes, including    orally, rectally, topical routes, including nasal, intranasal, and    other routes, including epidermal or patch delivery.-   22. Use according to any of the preceding embodiments, wherein the    non-chronic or chronic inflammatory diseases are selected from    diseases of the gastrointestinal tract including diseases of the    esophagus, stomach, first, second and third part of the duodenum,    jejunum, ileum, the ileo-cecal complex, large intestine, of the    ascending, transverse and descending colon sigmoid colon and rectum,    chronic inflammatory digestive diseases, characterized by an    inflammation of the colon, including colitis, selected from Colitis    ulcerosa (ulcerative colitis), Morbus Crohn (Crohn's disease),    diversion colitis, ischemic colitis, infectious colitis, fulminant    colitis, chemical colitis, microscopic colitis, lymphocytic colitis,    collageneous colitis, indeterminate colitis and atypical colitis.

The invention claimed is:
 1. A method of treating Crohn's disease in amammalian subject, the method comprising administering a pharmaceuticalcomposition to the subject in need of treatment thereof, the compositioncomprising a c-Jun N-terminal kinase (JNK) specific inhibitor consistingof having an amino acid sequence with at least 95% sequence identity toSEQ ID NO:
 11. 2. The method of claim 1, wherein the JNK inhibitor bindsJNK.
 3. The method of claim 1, wherein the JNK inhibitor inhibits theactivation of at least one JNK targeted transcription factor when theJNK inhibitor is present in a JNK expressing cell.
 4. The method ofclaim 3, wherein the JNK targeted transcription factor is selected fromthe group consisting of c-Jun, ATF2, and Elk1.
 5. The method of claim 1,wherein the JNK inhibitor alters a JNK effect when the peptide ispresent in a JNK expressing cell.
 6. The method of claim 1, wherein theJNK inhibitor is composed of D-amino acids.
 7. The method of claim 1,wherein the pharmaceutical composition is to be administered byintravenous administration.
 8. The method of claim 1, wherein the JNKinhibitor comprises a JNK binding domain that binds JNK.
 9. The methodof claim 1, wherein the JNK inhibitor comprises a JNK binding domainthat inhibits the activation of at least one JNK targeted transcriptionfactor when the JNK inhibitor is present in a JNK expressing cell. 10.The method of claim 9, wherein the JNK targeted transcription factor isselected from the group consisting of c-Jun, ATF2, and Elk1.
 11. Themethod of claim 1, wherein the JNK inhibitor alters a JNK effect whenthe amino acid sequence is present in a JNK expressing cell.